The Analgesic Effect Of Dipyrone In Peripheral Tissue Involves Two Different Mechanisms: Neuronal K(atp) Channel Opening And Cb(1) Receptor Activation.

Dipyrone (metamizole) is an analgesic pro-drug used to control moderate pain. It is metabolized in two major bioactive metabolites: 4-methylaminoantipyrine (4-MAA) and 4-aminoantipyrine (4-AA). The aim of this study was to investigate the participation of peripheral CB1 and CB2 cannabinoid receptors activation in the anti-hyperalgesic effect of dipyrone, 4-MAA or 4-AA. PGE2 (100ng/50µL/paw) was locally administered in the hindpaw of male Wistar rats, and the mechanical nociceptive threshold was quantified by electronic von Frey test, before and 3h after its injection. Dipyrone, 4-MAA or 4-AA was administered 30min before the von Frey test. The selective CB1 receptor antagonist AM251, CB2 receptor antagonist AM630, cGMP inhibitor ODQ or KATP channel blocker glibenclamide were administered 30min before dipyrone, 4-MAA or 4-AA. The antisense-ODN against CB1 receptor expression was intrathecally administered once a day during four consecutive days. PGE2-induced mechanical hyperalgesia was inhibited by dipyrone, 4-MAA, and 4-AA in a dose-response manner. AM251 or ODN anti-sense against neuronal CB1 receptor, but not AM630, reversed the anti-hyperalgesic effect mediated by 4-AA, but not by dipyrone or 4-MAA. On the other hand, the anti-hyperalgesic effect of dipyrone or 4-MAA was reversed by glibenclamide or ODQ. These results suggest that the activation of neuronal CB1, but not CB2 receptor, in peripheral tissue is involved in the anti-hyperalgesic effect of 4-aminoantipyrine. In addition, 4-methylaminoantipyrine mediates the anti-hyperalgesic effect by cGMP activation and KATP opening.

Chronic ethanol intake modulates vascular levels of endothelin-1 receptor and enhances the pressor response to endothelin-1 in anaesthetized rats

Background and purpose: The contribution of endothelin-1 (ET-1) to vascular hyper-reactivity associated with chronic ethanol intake, a major risk factor in several cardiovascular diseases, remains to be investigated. Experimental approach: The biphasic haemodynamic responses to ET-1 (0.01-0.1 nmol kg(-1), i.v.) or to the selective ET(B) agonist, IRL1620 (0.001-1.0 nmol kg(-1), i.v.), with or without ET(A) or ET(B) antagonists (BQ123 (c(DTrp-Dasp-Pro-Dval-Leu)) at 1 and 2.5 mg kg(-1) and BQ788 (N-cis-2,6-dimethyl-piperidinocarbonyl-L-gamma-methylleucyl1-D-1methoxycarbonyltryptophanyl-D-norleucine) at 0.25 mg kg(-1), respectively) were tested in anaesthetized rats, after 2 weeks` chronic ethanol treatment. Hepatic parameters and ET receptor protein levels were also determined. Key results: The initial hypotensive responses to ET-1 or IRL1620 were unaffected by chronic ethanol intake, whereas the subsequent pressor effects induced by ET-1, but not by IRL1620, were potentiated. BQ123 at 2.5 but not 1 mg kg(-1) reduced the pressor responses to ET-1 in ethanol-treated rats. Conversely, BQ788 (0.25 mg kg(-1)) potentiated ET-1-induced increases in mean arterial blood pressure in control as well as in ethanol-treated rats. Interestingly, in the latter group, increases in heart rate, induced by ET-1 at a dose of 0.025 mg kg(-1) were enhanced following ET(B) receptor blockade. Finally, we observed higher levels of ET(A) receptor in the heart and mesenteric artery and a reduction of ET(B) receptor protein levels in the aorta and kidney from rats chronically treated with ethanol. Conclusions and implications: Increased vascular reactivity to ET-1 and altered protein levels of ET(A) and ET(B) receptors could play a role in the pathogenesis of cardiovascular complications associated with chronic ethanol consumption.

Ameloblast apoptosis and IGF-1 receptor expression in the continuously erupting rat incisor model

Enamel-producing cells (ameloblasts) pass through several phenotypic and functional stages during enamel formation. In the transition between secretory and maturation stages, about one quarter of the ameloblasts suddenly undergo apoptosis. We have studied this phenomenon using the continuously erupting rat incisor model. A special feature of this model is that all stages of ameloblast differentiation are presented within a single longitudinal section of the developing tooth. This permits investigation of the temporal sequence of gene and growth factor receptor expression during ameloblast differentiation and apoptosis. We describe the light and electron microscopic morphology of ameloblast apoptosis and the pattern of insulin-like growth factor-1 receptor expression by ameloblasts in the continuously erupting rat incisor model. In the developing rat incisor, ameloblast apoptosis is associated with downregulated expression of the insulin-like growth factor-1 receptor. These data are consistent with the hypothesis that ameloblasts are hard wired for apoptosis and that insulin-like growth factor-1 receptor expression is required to block the default apoptotic pathway. Possible mechanisms of insulin-like growth factor-1 inhibition of ameloblast apoptosis are presented. The rat incisor model may be useful in studies of physiological apoptosis as it presents apoptosis in a predictable pattern in adult tissues.

Maternal and neonatal interleukin-1 receptor antagonist genotype and pregnancy outcome in a population with a high rate of pre-term birth

Problem We evaluated associations between a length polymorphism in intron 2 of the gene coding for IL-1ra (gene symbol IL1RN) and pregnancy outcome in a population with a high rate of preterm birth. Method of study Subjects were pregnant women in Maceio, Brazil and their newborns. DNA was tested for IL1RN genotypes and alleles by gene amplification using primer pairs that spanned the polymorphic region. Every subject completed a detailed questionnaire. Results The frequency of allele 2 (IL1RN*2) carriage was elevated in mothers with a spontaneous preterm birth (SPTB) in the current pregnancy (P = 0.02) and also with a prior preterm delivery (P = .01). Both SPTB with intact membranes (P = 0.01) and SPTB preceded by pre-term pre-mature rupture of membranes (P = .03) were associated with IL1RN*2 carriage. A previous fetal demise was more than twice as prevalent in mothers positive for two copies of IL1RN*2. Conclusion Maternal carriage of IL1RN*2 increases susceptibility to inflammation-triggered spontaneous pre-term birth.

Regulation of CSF-1 receptor expression

Cells of the mononuclear phagocyte lineage possess receptors for macrophage colony-stimulating factor (CSF-1) encoded by the c-fms protooncogene and respond to CSF-1 with increased survival, growth, differentiation, and reversible changes in function. The c-fms gene is itself a macrophage differentiation marker. In whole mount analyses of mRNA expression in embryos, c-fms is expressed at very high levels on placental trophoblasts. It is detectable on individual cells in the yolk sac around 8.5 to 9 days postcoitus, appears on isolated cells in the head of the embryo around 9.5 dpc, and appears on numerous cells throughout the embryo by day 10.5. The extent of c-fms expression is much greater than for other macrophage-specific genes including lysozyme and a macrophage-specific protein tyrosine phosphatase. Our studies of the cis-acting elements of the c-fms promoter have indicated a key role for collaboration between the macrophage-specific transcription factor, Pu.1, which functions in determining the site of transcription initiation, and other members of the Ets transcription factor family. This is emerging as a common pattern in macrophage-specific promoters. We have shown that two PU box elements alone can function as a macrophage-specific promoter. The activity of both the artifical promoter and the c-fms promoter is activated synergistically by coexpression of Pu.1 and another Ets factor, c-Ets-2. A 3.5kb c-fms exon 2 promoter (but not the 300bp proximal promoter) is also active in a wide diversity of tumor cell lines. The interesting exception is the melanoma cell line K1735, in which the promoter is completely shut down and expression of c-fms causes growth arrest and cell death. The activity of the exon 2 promoter in these nonmacrophages is at least as serum responsive as the classic serum-responsive promoter of the c-fos gene. It is further inducible in nonmacrophages by coexpression of the c-fms product. Unlike other CSF-1/c-fms-responsive promoters, the c-fms promoter is not responsive to activated Ras even when c-Ets-2 is coexpressed. In most lines, production of full length c-fms is prevented by a downstream intronic terminator, but in Lewis lung carcinoma, read-through does occur, and expression of both c-fms and other macrophage-specific genes such as lysozyme and urokinase becomes detectable in conditions of serum deprivation. (C) 1997 Wiley-Liss, Inc.

Interleukin-1 receptor antagonist gene (IL1RN) polymorphism possibly associated to severity of rheumatic carditis in a Brazilian cohort

Aims: To evaluate the IL1RN polymorphism as a possible marker for Rheumatic Fever (RF) susceptibility or disease severity. Methods: The genotypes of 84 RF patients (Jones criteria) and 84 normal race-matched controls were determined through the analysis of the number of 86-bp tandem repeats in the second intron of IL1RN. The DNA was extracted from peripheral-blood leukocytes and amplified with specific primers. Clinical manifestations of RF were obtained through a standardized questionnaire and an extensive chart review. Carditis was defined as new onset cardiac murmur that was perceived by a trained physician with corresponding valvae regurgitation or stenosis on echocardiogram. Carditis was classified as severe in the presence of congestive heart failure or upon the indication for cardiac surgery. The statistical association among the genotypes, RF and its clinical variations was determined. Results: The presence of allele I and the genotype A1/A1 were found less frequently among patients with severe carditis when compared to patients without this manifestation (OR = 0.11, p = 0.031; OR = 0.092, p = 0.017). Neither allele I nor allele 2 were associated with the presence of RF (p = 0.188 and p = 0.106), overall carditis (p = 0.578 and p = 0.767), polyarthritis (p = 0.343 and p = 0.313) and chorea (p = 0.654 and p = 0.633). Conclusion: In the Brazilian population, the polymorphism of the IL-1ra gene is a relevant factor for rheumatic heart disease severity. (C) 2009 Elsevier Ltd. All rights reserved.

Topical interleukin-1 receptor antagonist inhibits inflammatory cell infiltration into the cornea

Interleukin (IL)-1 alpha and beta are important modulators of many functions of corneal epithelial and stromal cells that occur following injury to the cornea, including the influx of bone marrow-derived inflammatory cells into the stroma attracted by chemokines released from the stroma and epithelium. In this study, we examined the effect of topical soluble IL-1 receptor antagonist on bone marrow-derived cell influx following corneal epithelial scrape injury in a mouse model. C57BL/6 mice underwent corneal epithelial scrape followed by application of IL-1 receptor antagonist (Amgen, Thousand Oaks, CA) at a concentration of 20 mg/ml or vehicle for 24 h prior to immunocytochemical detection of marker CD11b-positive cells into the stroma. In two experiments, topical IL-1 receptor antagonist had a marked effect in blocking cell influx. For example, in experiment 1, topical IL-1 receptor antagonist markedly reduced detectible CD11b-positive cells into the corneal stroma at 24 It after epithelial injury compared with the vehicle control (3.5 +/- 0.5 (standard error of the mean) cells/400x field and 13.9 +/- 1.2 cells/400x field, respectively, p < 0.01). A second experiment with a different observer performing cell counting had the same result. Thus, the data demonstrate conclusively that topical IL-1 receptor antagonist markedly down-regulates CD-11b-positive monocytic cell appearance in the corneal stroma. Topical IL-1 receptor antagonist could be an effective adjuvant for clinical treatment of corneal conditions in which unwanted inflammation has a role in the pathophysiology of the disorder. (c) 2008 Elsevier Ltd. All rights reserved.

A Role for the Substance P/NK-1 Receptor Complex in Cell Proliferation in Oral Squamous Cell Carcinoma

Objective: To investigate the presence and distribution of substance P (SP) and neurokinin I receptor (NK-IR) in oral squamous cell carcinoma (OSCC) and their relationship with proliferation. Patients and Methods: Ninety OSCCs from 73 patients were immunohistochemically analyzed using monoclonal antibodies against SP, NK-IR and Ki-67 in a case and control study. Results: Seventy-one percent (n=49) of cases expressed SP on tumour cell membrane, 81.3% (n=69) in cytoplasm, 39.4% (n=28) in nucleus, 81.6% (n=71) in infiltrating lymphocytes, and 58.1% (n=43) in peritumoural or intratumoural blood vessels; 14% (n=12) of cases expressed NK-1R on tumour cell membrane, 50% (n=43) in cytoplasm, 48.3% (n=42) in infiltrating lymphocytes and 22.5% (n=18) in tumour blood vessels. All cases expressed Ki-67, which was expressed in >25% of tumour cells in 79.8% of cases (n=63). Direct significant associations were observed in SP expression between different tissue levels (p<0.01), between SP and NK-IR tumour cell membrane expression (p<0.01), and between joint,SP and NK-IR expression in tumour cell cytoplasm and a higher expression of Ki-67 (p<0.05). Conclusion: The ubiquitous presence of SP strongly suggests a role for SP/NK-1R complex in tumour development and progression and possibly for NK-IR antagonists, such as L-773060, in the management of patients with oral cancer.

Melanocortin-1 receptor genotype is a risk factor for basal and squamous cell carcinoma

MC1R gene variants have previously been associated with red hair and fair skin color, moreover skin ultraviolet sensitivity and a strong association with melanoma has been demonstrated for three variant alleles that are active in influencing pigmentation: Arg151Cys, Arg160Trp, and Asp294His. This study has confirmed these pigmentary associations with MC1R genotype in a collection of 220 individuals drawn from the Nambour community in Queensland, Australia, 111 of whom were at high risk and 109 at low risk of basal cell squamous cell carcinoma. Comparative allele frequencies for nine MC1R variants that have been reported in the Caucasian population were determined for these two groups, and an association between prevalence of basal cell carcinoma, squamous cell carcinoma, solar keratosis and the same three active MC1R variant alleles was demonstrated [odds ratio=3.15 95% CI (1.7, 5.82)]. Three other commonly occurring variant alleles: Val60Leu, Val92Met, and Arg163Gln were identified as having a minimal impact on pigmentation phenotype as well as basal cell carcinoma and squamous cell carcinoma risk. A significant heterozygote effect was demonstrated where individuals carrying a single MC1R variant allele were more likely to have fair and sun sensitive skin as well as carriage of a solar lesion when compared with those individuals with a consensus MC1R genotype. After adjusting for the effects of pigmentation on the association between MC1R variant alleles and basal cell carcinoma and squamous cell carcinoma risk, the association persisted, confirming that presence of at least one variant allele remains informative in terms of predicting risk for developing a solar-induced skin lesion beyond that information gained through observation of pigmentation phenotype.

Growth hormone receptor and IGF-1 receptor immunoreactivity during orthodontic tooth movement in the prednisolone-treated rat

Bone remodeling during tooth movement is regulated by local and systemic factors. Two regulators of bone metabolism are growth hormone (GH) and insulin-like growth factor-I (IGF-1). Their effects are mediated via binding to GH receptor (GHR) and IGF-I receptor (IGF-IR) in target tissues. Corticosteroids may affect the activity of these growth factors. This study examined the effect of prednisolone on GHR and IGF-IR expression in dental tissues following orthodontic tooth movement. The corti ticosteroid-treated group (N = 6) was administered prednisolone ( 1 mg/kg,) daily and the control group (N = 6) received equivalent volumes of saline. An orthodontic force (30 g) was applied to the maxillary first molar. Animals were sacrificed 12 days postappliance insertion. Sagittal sections of the first molar were stained for GHR and IGF-IR immunoreactivity. GHR and IGF-IR cell counts were elevated following appliance-treatment. Orthodontic tooth movement appeared to up-regulate GHR and IGF-IR immunoreactivity, but this up-regulation was reduced following prednisolone treatment. The suppression of GHR and IGF-I immunoreactivity in steroid-treated animals infers the mechanism whereby bone resorption and deposition, necessary for orthodontic tooth movement, may be inhibited by prednisolone. However, at 12 days postappliance insertion. no difference in orthodontic tooth movement was observed following low-dose prednisolone treatment.

The human melanocortin-1 receptor locus: analysis of transcription unit, locus polymorphism and haplotype evolution

The complete sequence of the MCIR locus has been assembled, the coding region of the gene is intronless and placed within a 12 kb region flanked by the NULP1 and TUBB4 genes. The immediate promoter region has an E-box site with homology to the M-box consensus known to bind the microphthalmia transcription factor (MITF), however, promoter deletion analysis and transactivation studies have failed to show activation through this element by MITF. Polymorphism within the coding region, immediate 5' promoter region and a variable number tandem repeat (VNTR) minisatellite within the locus have been examined in a collection of Caucasian families and African individuals. Haplotype analysis shows linkage disequilibrium between the VNTR and MCIR coding region red hair variant alleles which can be used to estimate the age of these missense changes. Assuming a mean VNTR mutation rate of 1% and a star phylogeny, we estimate the Arg151Cys variant arose 7500 years before the present day, suggesting these variants may have arisen in the Caucasian population more recently than previously thought. (C) 2001 Published by Elsevier Science B.V.

The therapeutic potential of endothelin-1 receptor antagonists and endothelin-converting enzyme inhibitors on the cardiovascular system

Clinical trials have established bosentan, an orally active non-selective endothelin (ET) receptor antagonist, as a beneficial treatment in pulmonary hypertension. Trials have also shown short-term benefits of bosentan in systemic hypertension and congestive heart failure. However, bosentan also increased plasma levels of ET-1, probably by inhibiting the clearance of ET-1 by endothelin type B (ET.) receptors, and this may mean its effectiveness is reduced with long-term clinical use. Preliminary data suggests that selective endothelin type A (ETA) receptor antagonists (BQ-123, sitaxsentan) may be more beneficial than the non-selective ET receptor antagonists in heart failure, especially when the failure is associated with pulmonary hypertension. Experimental evidence in animal disease models suggests that non-selective ET or selective ETA receptor antagonism may have a role in the treatment of athero-sclerosis, restenosis, myocarditis, shock and portal hypertension. In animal models of myocardial infarction and/or reperfusion injury, non-selective ET or selective ETA receptor antagonists have beneficial or detrimental effects depending on the conditions and agents used. Thus clinical trials of the nonselective ET or selective ETA receptor antagonists in these conditions are not presently warranted. Several selective endothelin-converting enzyme inhibitors tors have been synthesised recently, and these are only beginning to be tested in animal models of cardiovascular disease, and thus the clinical potential of these inhibitors is still to be defined.

The role of melanocortin-1 receptor polymorphism in skin cancer risk phenotypes

We have examined melanocortin-1 receptor (MC1R) variant allele frequencies in the general population and in a collection of adolescent dizygotic and monozygotic twins to determine statistical associations of pigmentation phenotypes with increased skin cancer risk. This included hair and skin color, freckling, mole count and sun exposed skin reflectance. Nine variants were studied and designated as either strong R (OR = 63; 95% CI 32-140) or weak r (OR = 5; 95% CI 3-11) red hair alleles. Penetrance of each MC1R variant allele was consistent with an allelic model where effects were multiplicative for red hair but additive for skin reflectance. To assess the interaction of the brown eye color gene BEY2/OCA2 on the phenotypic effects of variant MC1R alleles we imputed OCA2 genotype in the twin collection. A modifying effect of OCA2 on MC1R variant alleles was seen on constitutive skin color, freckling and mole count. In order to study the individual effects of these variants on pigmentation phenotype we have established a series of human primary melanocyte strains genotyped for the MC1R receptor. These include strains which are MC1R wild-type consensus, variant heterozygotes, and homozygotes for strong R alleles Arg151Cys and Arg160Trp. Ultrastructural analysis demonstrated that only consensus strains contained stage III and IV melanosomes in their terminal dendrites whereas Arg151Cys and Arg160Trp homozygous strains contained only immature stage I and II melanosomes. Such genetic association studies combined with the functional analysis of MC1R variant alleles in melanocytic cells should provide a link in understanding the association between pigmentary phototypes and skin cancer risk.

Screening of human primary melanocytes of defined melanocortin-1 receptor genotype: Pigmentation marker, ultrastructural and UV-survival studies

Recent population studies have demonstrated an association with the red-hair and fair-skin phenotype with variant alleles of the melanocortin-1 receptor (MC1R) which result in amino acid substitutions within the coding region leading to an altered receptor activity. In particular, Arg151Cys, Arg160Trp and Asp294His were the most commonly associated variants seen in the south-east Queensland population with at least one of these alleles found in 93% of those with red hair. In order to study the individual effects of these variants on melanocyte biology and melanocytic pigmentation, we established a series of human melanocyte strains genotyped for the MC1R receptor which included wild-type consensus, variant heterozygotes, compound heterozygotes and homozygotes for Arg151Cys, Arg160Trp, Val60Leu and Val92Met alleles. These strains ranged from darkly pigmented to amelanotic, with all strains of consensus sequence having dark pigmentation. UV sensitivity was found not to be associated with either MC1R genotype or the level of pigmentation with a range of sensitivities seen across all genotypes. Ultrastructural analysis demonstrated that while consensus strains contained stage IV melanosomes in their terminal dendrites, Arg151Cys and Arg160Trp homozygote strains contained only stage II melanosomes. This was despite being able to show expression of tyrosinase and tyrosinase-related protein-1 markers, although at reduced levels and an ability to convert exogenous 3,4-dihydroxyphenyl-alanine (DOPA) to melanin in these strains.