Cloning, characterization and central nervous system distribution of receptor activity modifying proteins in the rat.

Calcitonin gene-related peptide (CGRP), adrenomedullin (ADM), amylin and calcitonin (CT) are structurally and functionally related neuropeptides. It has recently been shown that the molecular pharmacology of CGRP and ADM is determined by coexpression of one of three receptor activity-modifying proteins (RAMPs) with calcitonin receptor-like receptor (CRLR). Furthermore, RAMP proteins have also been shown to govern the pharmacology of the calcitonin receptor, which in association with RAMP1 or RAMP3, binds amylin with high affinity. In this study, we have cloned the rat RAMP family and characterized the pharmacology of rat CGRP and ADM receptors. Rat RAMP1, RAMP2 and RAMP3 shared 72%, 69% and 85% homology with their respective human homologues. As expected CRLR-RAMP1 coexpression conferred sensitivity to CGRP, whilst association of RAMP2 or RAMP3 with CRLR conferred high affinity ADM binding. Using specific oligonucleotides we have determined the expression of RAMP1, RAMP2 and RAMP3 mRNAs in the rat central nervous system by in situ hybridization. The localization of RAMP mRNAs was heterogeneous. RAMP1 mRNA was predominantly expressed in cortex, caudate putamen and olfactory tubercles; RAMP2 mRNA was most abundant in hypothalamus; and RAMP3 was restrictively expressed in thalamic nuclei. Interestingly, in specific brain areas only a single RAMP mRNA was often detected, suggesting mutual exclusivity in expression. These data allow predictions to be made of where each RAMP protein may heterodimerize with its partner G-protein-coupled receptor(s) at the cellular level and consequently advance current understanding of cellular sites of action of CGRP, ADM, amylin and CT. Furthermore, these localization data suggest that the RAMP family may associate and modify the behaviour of other, as yet unidentified neurotransmitter receptors.

Targeted deletion of the A3 adenosine receptor confers resistance to myocardial ischemic injury and does not prevent early preconditioning.

We used mice with genetic disruption of the A3 adenosine receptor (AR) gene (A3AR(-/-)mice) to assess the in vivo role of the A3AR in modulating myocardial ischemia/reperfusion injury and preconditioning (PC). Surprisingly, infarct size induced by 30 min of coronary artery occlusion and 24 h of reperfusion was 35% smaller in A3AR(-/-)compared to wild-type mice (A3AR(+/+)). The reduction in infarct size was not the result of differences in heart rate, body temperature or increased cardiac expression of A1ARs. However, neutrophil infiltration within infarcted regions was less in A3AR(-/-)mice. Furthermore, ischemic PC induced by either a single episode (one 5 min occlusion) or multiple episodes (six 4 min occlusions) of ischemia produced equivalent reductions in infarct size in A3AR(-/-)and A3AR(+/+)mice. These results indicate that, in the mouse, (i) A3ARs play an injurious role during acute myocardial ischemia/reperfusion injury, possibly by exacerbating the inflammatory response, and (ii) A3ARs are not necessary for the development of the early phase of ischemic PC.

Adenosine and inosine increase cutaneous vasopermeability by activating A(3) receptors on mast cells.

Adenosine has potent effects on both the cardiovascular and immune systems. Exposure of tissues to adenosine results in increased vascular permeability and extravasation of serum proteins. The mechanism by which adenosine brings about these physiological changes is poorly defined. Using mice deficient in the A(3) adenosine receptor (A(3)AR), we show that increases in cutaneous vascular permeability observed after treatment with adenosine or its principal metabolite inosine are mediated through the A(3)AR. Adenosine fails to increase vascular permeability in mast cell-deficient mice, suggesting that this tissue response to adenosine is mast cell-dependent. Furthermore, this response is independent of activation of the high-affinity IgE receptor (FcepsilonR1) by antigen, as adenosine is equally effective in mediating these changes in FcepsilonR1 beta-chain-deficient mice. Together these results support a model in which adenosine and inosine induce changes in vascular permeability indirectly by activating mast cells, which in turn release vasoactive substances. The demonstration in vivo that adenosine, acting through a specific receptor, can provoke degranulation of this important tissue-based effector cell, independent of antigen activation of the high-affinity IgE receptor, supports an important role for this nucleoside in modifying the inflammatory response.

Disruption of the A(3) adenosine receptor gene in mice and its effect on stimulated inflammatory cells.

The A(3) adenosine receptor (A3AR) is one of four receptor subtypes for adenosine and is expressed in a broad spectrum of tissues. In order to study the function of A3AR, a mouse line carrying a mutant A(3) allele was generated. Mice homozygous for targeted disruption of the A3AR gene, A3AR(-/-), are fertile and visually and histologically indistinguishable from wild type mice. The lack of a functional receptor in the A3AR(-/-) mice was confirmed by molecular and pharmacological analyses. The absence of A3AR protein expression in the A3AR(-/-) mice was demonstrated by lack of N(6)-(4-amino-3-[(125)I]iodobenzyl)adenosine binding to bone marrow-derived mast cell membranes that were found to express high levels of A3AR in wild type mice. In A3AR(-/-) mice, the density of A(1) and A(2A) adenosine receptor subtypes was the same as in A3AR(+/+) mice as determined by radioligand binding to brain membranes. Additionally, A(2B) receptor transcript expression was not affected by ablation of the A3AR gene. A3AR(-/-) mice have basal heart rates and arterial blood pressures indistinguishable from A3AR(+/+) mice. Functionally, in contrast to wild type mice, adenosine and the A3AR-specific agonist, 2-chloro-N(6)-(3-iodobenzyl)-adenosine-5'-N-methyl-carboxamide (2-Cl-IB-MECA), elicit no potentiation of antigen-dependent degranulation of bone marrow-derived mast cells from A3AR(-/-) mice as measured by hexosaminidase release. Also, the ability of 2Cl-IB-MECA to inhibit lipopolysaccharide-induced tumor necrosis factor-alpha production in vivo was decreased in A3AR(-/-) mice in comparison to A3AR(+/+) mice. The A(2A) adenosine receptor agonist, 2-p-(2-carboxyethyl)phenylamino)-5'-N-ethylcarboxamidoadenosine, produced inhibition of lipopolysaccharide-stimulated tumor necrosis factor-alpha production in both A3AR(-/-) and A3AR(+/+) mice. These results show that the inhibition in vivo can be mediated by multiple subtypes, specifically the A(3) and A(2A) adenosine receptors, and A3AR activation plays an important role in both pro- and anti-inflammatory responses.

Adenosine A3 receptor expression and function in eosinophils.

The A3 adenosine receptor is widely expressed in human tissues with the most abundant expression in the lung and liver, but the predominant cellular localization and functions of this receptor in humans are unknown. Since adenosine influences the activation of circulating and resident inflammatory cells within the lung and leads to exaggerated airway narrowing in individuals with inflammatory airway disorders, we hypothesized that A3 receptor gene expression is localized to inflammatory cells and that gene expression is upregulated in airway inflammation. Lung and airway tissue were obtained at thoracotomy from nonsmoking subjects and subjects with inflammatory airway disorders associated with tobacco smoke or asthma. In situ hybridization identified A3 receptors in mesenchymal cells and eosinophils within the lamina propria of the airways and the adventitia of blood vessels, but not in mast cells. A3 receptor transcripts were highly expressed in peripheral blood eosinophils purified from atopic donors (6.36 +/- 0.60 pg/microg total RNA) in comparison with neutrophils (0.26 +/- 0.06 pg/microg) or mononuclear cells (0.9 +/- 0.15 pg/microg). Mean A3 receptor transcript abundance was greater in lung tissue from subjects with airway inflammation (0.33 +/- 0.04 pg/microg total RNA) than in normal lung (0.24 +/- 0.03 pg/microg total RNA, P = 0.035). The A3 receptor agonist N6-(4-amino-3-iodobenzyl)adenosine dose-dependently inhibited platelet activating factor-induced eosinophil chemotaxis to a maximum of 41%. This inhibitory effect was completely abolished by addition of the A3 receptor selective antagonist 3-(3-iodo-4-aminobenzyl)-8-(4-oxyacetate)phenyl-1-propylxanthine. We conclude that A3 receptors are primarily expressed on eosinophils in human lung, where they mediate inhibition of eosinophil chemotaxis. Specific A3 receptor ligands may be useful agents in the treatment of eosinophil-dependent diseases such as asthma and rhinitis.

Cloning and chromosomal localization of the human A2b adenosine receptor gene (ADORA2B) and its pseudogene.

To determine the chromosomal localization of the human A2b adenosine receptor, the corresponding genomic clone was isolated and used as a probe for fluorescence in situ hybridization to metaphase chromosomes. Partial sequence analysis of the A2b gene (AD-ORA2B) revealed an intron that interrupted the coding region corresponding to the second intracellular loop similar to that reported for A1 and A2a adenosine receptor genes. A pseudogene for the A2b receptor was also identified; it exhibited 79% identity to the A2b adenosine receptor cDNA coding sequence and contained multiple deletions, point mutations, and frame shifts and two in-frame stops. These changes would result in the inability to encode a functional receptor. The genomic clones were utilized to localize the A2b receptor to chromosome 17p12 and the A2b pseudogene to chromosome 1q32.

Molecular cloning and characterization of the human A3 adenosine receptor.

The human A3 adenosine receptor was cloned from a striatal cDNA library using a probe derived from the homologous rat sequence. The cDNA encodes a protein of 318 amino acids and exhibits 72% and 85% overall identity with the rat and sheep A3 adenosine receptor sequences, respectively. Specific and saturable binding of the adenosine receptor agonist N6-(4-amino-3-[125I]iodobenzyl)adenosine [125I]ABA was measured on the human A3 receptor stably expressed in Chinese hamster ovary cells with a Kd = 10 nM. The potency order for adenosine receptor agonists was N-ethylcarboxamidoadenosine (NECA) > or = (R)-N6-phenyl-2-propyladenosine [(R)-PIA] > N6-cyclopentyladenosine (CPA) > (S)-N6-phenyl-2-propyladenosine [(S)-PIA]. The human receptor was blocked by xanthine antagonists, most potently by 3-(3-iodo-4-aminobenzyl)-8-(4-oxyacetate)phenyl-1-propylxanthine (I-ABOPX) with a potency order of I-ABOPX > 1,3-dipropyl-8-(4-acrylate)phenylxanthine > or = xanthine amino congener >> 1,3-dipropyl-8-cyclopentylxanthine. Adenosine, NECA, (R)- and (S)-PIA, and CPA inhibited forskolin-stimulated cAMP accumulation by 30-40% in stably transfected cells; I-ABA is a partial agonist. When measured in the presence of antagonists, the dose-response curves of NECA-induced inhibition of forskolin-stimulated cAMP accumulation were right-shifted. Antagonist potencies determined by Schild analyses correlated well with those established by competition for radioligand binding. The A3 adenosine receptor transcript is widespread and, in contrast to the A1, A2a, and A2b transcripts, the most abundant expression is found in the lung and liver. The tissue distribution of A3 mRNA is more similar to the widespread profile found in sheep than to the restricted profile found in the rat. This raises the possibility that numerous physiological effects of adenosine may be mediated by A3 adenosine receptors.

Ovarian histology and function after total abdominal hysterectomy.

Ovarian histology and function were assessed before and after total abdominal hysterectomy in 25 patients with symptomatic uterine myomata. Immediately before hysterectomy, bilateral ovarian biopsies were performed, and, 12 months later, all patients underwent a second ovarian biopsy through laparoscopy. Histologic study of the ovaries one year after total abdominal hysterectomy showed stromal cell hyperplasia, thickening of the tunica albuginea, and a significant decrease of follicular reserve, follicular cysts, and corpora albicantia. There was no significant difference in the number of atretic follicles and corpora lutea. The serum levels of all hormones studied were unchanged 12 months after the surgical procedures.

Reconstrucao microcirurgica das trompas. Experiencia com 24 casos. / Microsurgical reconstruction of the fallopian tubes. Experience with 24 cases

A cirurgia reconstrutora das trompas ganhou grande enfase com a introducao em clinica das tecnicas de microcirurgia. Estas tecnicas, que incluem nao somente aumento optico, mas abrangem outros principios, como manipulacao delicada das estruturas locais com trauma tecidual minimo, hemostasia cuidadosa, emprego de material inerte e resseccao cuidadosa das areas lesadas, aumentaram os indices de sucessos nos casos de recenalizacao das trompas. No presente estudo, os autores apresentam sua experiencia com 24 pacientes submetidos a reconstrucao tubaria com tecnica microcirurgica. Neste material observaram-se indices de permeabilidade tubaria e de gravidez de 80% e 53% respectivamente, com resultados piores nas obstrucoes de origem inflamatoria. Consideracoes sobre os casos estudados e sobre os fatores que interferem com os resultados deste procedimento cirurgico sao apresentados pelos autores

Estudo clinico e laboratorial de pacientes climatericas (fase pos-menopausica) submetidas ao LIR-1660. / Clinical and laboratorial study of climacteric patients (post-menopausal phase) submitted to LIR 1660

Trinta pacientes na pos-menopausa fisiologica foram tratadas com LIR-1660 na dosagem de 100 mg/dia durante 20 dias. Atraves do indice menopausal de Kupperman, da dosagem de FSH, LH prolactina, estradiol e da colpocitologia hormonal observaram-se as modificacoes apos o tratamento. Pode-se concluir ser a droga eficaz no controle dos sintomas neurovegetativos, mas nao determina alteracoes nas concentracoes sanguineas dos hormonios estudados e na colpocitologia hormonal. Em nenhum caso foi observado efeitos colaterais e galactorreia

Estudo clinico de nova medicacao (LIR-1660) na sindrome climaterica (fase pos-menopausica) / Clinical study of a new drug (LTR-1660) in the climateric syndrome (post-menopausal phase)

Os autores estudaram 20 pacientes climatericas na fase pos-menopausica fisiologica, pertencentes a Secao de Climaterio da Clinica Ginecologica da FMUSP (Hospital das Clinicas - Servico do Prof. Carlos Alberto Salvatore), cujas idades oscilaram de 44 a 60 anos. A estas pacientes foi administrado LIR-1660 na dosagem de 100 mg por dia, via oral, por 20 dias consecutivos. Os resultados relacionados aos sintomas vasomotores foram satisfatorios em 80% dos casos e regulares em 20%; em nenhum caso observou-se efeito colateral a droga. Concluem os autores da eficacia desta substancia, e que a mesma representa uma boa alternativa terapeutica nas pacientes climatericas

Efeito do danazol na endometriose pelvica. Analise de 20 casos com seguimento de cinco anos. / Effect of danazol on pelvic endometriosis. Analysis of 20 cases with follow-up for 5 years

Os autores analisaram a eficacia do danazol, administrado por via oral nas doses de 400 mg e 600 mg/dia em pacientes inferteis com endometriose pelvica. Os sintomas mais comuns foram dismenorreia (55,0%) algia pelvica cronica (40,0%) e tumor anexial (30,0%). A amenorreia farmacologica e de importante valor prognostico, traduzindo a eficacia do tratamento. No esquema de 400 mg/dia, o indice global de resposta sintomatologica foi de 87,5%; de 600 mg/ dia, de 75.0%. Obteve-se gestacao em 9 (47,4%) das 19 enfermas desejosas de engravidar e o intervalo de tempo medio entre o termino da terapeutica e a gravidez foi de 2,9 meses. Por fim, a ocorrencia de gestacao em pacientes com endometriose pelvica, nesta casuistica, nao guardou relacao com o tempo de esterilidade previa.O periodo de seguimento foi de cinco anos em todos os casos

Medida vascular no carcinoma da mama.Estudo mamografico quantitativo. / Vascular measurement in brest cancer Quantitative mammographic study

O padrao vascular das mamas apreciado pela mamografia foi estudado quantitativamente entre 1978 e 1982. Assim, a vascularizacao mamaria em 10 portadoras de carcinoma (Grupo A) foi comparada com a existente em 10 pacientes com displasia (Grupo B). A idade media foi de 47,1 e 45 anos para os grupos A e B, respectivamente. O total de mensuracoes nas 40 mamas foi de 4.000, escolhidas ao acaso. O diametro medio dos vasos nas mamas carcinomatosas foi de 1,60 +/- 0,11 milimetros, ao passo que o da glandula contralateral foi de 0,72 +/- 0, 12 milimetros, dados estatisticamente diferentes. O diametro medio dos vasos nas pacientes com displasia apresentou-se estatisticamente igual nas glandulas direita a esquerda, isto e, 0,71 +/- 0,08 e 0,68 +/- 0,10 milimetros, respectivamente. Comparou-se o diametro medio dos vasos nas mamas das enfermas com displasia (0,69 +/- 0,09 milimetros) com o correspondente das 10 glandulas normais em portadoras de carcinoma (0,73 +/- 0,11 milimetros).Nao houve diferenca estatistica nos dados encontrados, sugerindo que o aumento da vascularizacao em mamas carcinomatosas resulta de fator local

Acao antimicrobiana local da associacao anfotericina B e cloridrato de tetraciclina em pacientes portadoras de leucorreia de diferentes etiologias. / Local antimicrobial action of the association of amphotericin B and tetracyclin in patients with leukorrhea of different etiologies

O objetivo do presente estudo e avaliar a eficacia da associacao de cloridrato de tetraciclina 100 mg e anfotericina B 50 mg na forma de creme vaginal em pacientes portadoras de corrimento vaginal. Neste sentido selecionamos no ambulatorio de ginecologia do Hospital das Clinicas da Faculdade de Medicina da Universidade de Sao Paulo (Servico do Prof. Carlos Alberto Salvatore), 40 pacientes, cujas idades variaram de 18 a 56 anos (media etaria 31 anos) com corrimento de aspecto inespecifico; 28 destas apresentaram dispareunia; 23 prurido; 22 ardor e disuria.A estas pacientes foi administrada esta associacao sob a forma de creme vaginal, um aplicador por dia, durante sete dias. Nas pacientes que permaneciam com qualquer sintoma, apos este intervalo, o tratamento era prolongado por mais sete dias. Antes do inicio do tratamento e sete dias apos o termino, foram realizados os seguintes exames: exame a fresco com hidroxido de potassio a 10% coloracao de Gram, Giemsa e Papanicolaou para detectar-se flora Gram-negativa, Candida, Trichomonas, Mycoplasma, Chlamydia, Neisseria e Hemophilus

Frozen section biopsy of ovarian neoplasms.

In a study of the frozen section biopsy of 120 ovarian neoplasms, correct results were obtained in 94.16%, incorrect in 4.16%, and inconclusive in 1.66% of the 120 cases. The "incorrect" results were all "false-negative." No inverse "false-positive" results were recorded, and the "incorrect" results occurred in cases of encapsulated neoplasms of a macroscopically benign appearance with small areas of malignant changes detected only by paraffin block technique. The authors conclude that frozen section biopsy of ovarian neoplasm is plausible so long as a pathologist is available who can be provided with the proper equipment during the surgery and that the technique is more indicated in patients up to the age of 40 because of the necessity of conservative management in this age group.

Reconstrucao da vulva, perineo e regiao perianal por retalho miocutaneo bilateral do musculo gracilis. / Reconstruction of the vulva, perineum and perianal region with myocutaneous flap of gracilis muscle

Os autores apresentam caso de reparacao da vulva, perineo e regiao perianal, pos ressecao de extensa area de radiodermite com enfermidade de Paget, mediante emprego de retalho miocutaneo bilateral do musculo gracilis, com resultado satisfatorio, de vez que nao houve estenose dos orificios naturais da regiao, alem da boa qualidade do revestimento cutaneo, apesar de ter ocorrido perda parcial de um dos retalhos