RESUMO
BACKGROUND: The effect of sarcopenia based on the total psoas muscle area (TPMA) on CT is inconclusive in patients undergoing abdominal aortic aneurysm (AAA) intervention. The aim of this prospective cohort study was to evaluate morphometric sarcopenia as a method of risk stratification in patients undergoing elective AAA intervention. METHODS: TPMA was measured on preintervention CT images of patients undergoing elective endovascular aneurysm repair (EVAR) or open aneurysm repair. Mortality was assessed in relation to preintervention TPMA using Cox regression analysis, with calculation of hazard ratios at 30 days, 1 year and 4 years. Postintervention morbidity was evaluated in terms of postintervention care, duration of hospital stay and 30-day readmission. Changes in TPMA on surveillance EVAR imaging were also evaluated. RESULTS: In total, 382 patient images acquired between March 2008 and December 2016 were analysed. There were no significant intraobserver and interobserver differences in measurements of TPMA. Preintervention TPMA failed to predict morbidity and mortality at all time points. The mean(s.d.) interval between preintervention and surveillance imaging was 361·3(111·2) days. A significant reduction in TPMA was observed in men on surveillance imaging after EVAR (mean reduction 0·63(1·43) cm2 per m2 ; P < 0·001). However, this was not associated with mortality (adjusted hazard ratio 1·00, 95 per cent c.i. 0·99 to 1·01; P = 0·935). CONCLUSION: TPMA is not a suitable risk stratification tool for patients undergoing effective intervention for AAA.
Assuntos
Aneurisma da Aorta Abdominal/cirurgia , Angiografia por Tomografia Computadorizada/métodos , Procedimentos Cirúrgicos Eletivos/métodos , Procedimentos Endovasculares/métodos , Músculos Psoas/diagnóstico por imagem , Sarcopenia/diagnóstico por imagem , Idoso , Aneurisma da Aorta Abdominal/diagnóstico por imagem , Aneurisma da Aorta Abdominal/mortalidade , Estudos de Coortes , Procedimentos Cirúrgicos Eletivos/mortalidade , Procedimentos Endovasculares/mortalidade , Feminino , Mortalidade Hospitalar/tendências , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prognóstico , Modelos de Riscos Proporcionais , Estudos Prospectivos , Análise de Regressão , Análise de Sobrevida , Resultado do TratamentoRESUMO
This article describes the physiological changes that take place during anaphylaxis, explores the ABCDE (airway, breathing, circulation, disability, exposure) approach to patient assessment and discusses the pharmacological interventions required to treat anaphylaxis effectively.
Assuntos
Anafilaxia/diagnóstico , Anafilaxia/terapia , Anafilaxia/enfermagem , Anafilaxia/fisiopatologia , Humanos , Avaliação em EnfermagemRESUMO
OBJECTIVE: To explore self-inflicted retinal burns from laser pointers in children. METHODS: Literature review of laser pointer retinal injuries in childhood and online survey of UK Consultant Ophthalmologists. A cohort of local children with self-inflicted injury is described. The matter is topical. We review progress in recent legislation and policy change in the UK. RESULTS: Four of 77 case reports of laser burns in childhood analysed reported psychological or behavioural issues. Three of four children in our cohort had such issues. Delay in diagnosis occurred in two of our patients. Structural retinal damage persisted for over 12 months in all four children (seven eyes). Our survey of UK ophthalmologists found 159 cases of injury (85% male), 80% under 20 years of age. The majority of the laser pointers were purchased online. Many patients (36%) suffered moderate vision loss (6/18 to 6/60 Snellen), while 17% (at least 11 patients) suffered severe vision loss (<6/60 Snellen). CONCLUSION: We highlight the risk of macular damage and vision loss from handheld lasers specifically in children with behavioural, learning or mental health issues. The diagnosis may be difficult or delayed in such children. In children with uncertain macular changes, ophthalmologists should explore the history for possible instances of exposure to handheld lasers pointers. Regulatory authorities and manufacturers of handheld lasers need to be aware of the risk to children. Furthermore, there is a need to better inform parents, carers and teachers of the risk of ocular self-injury from such lasers pointers.
Assuntos
Qualidade de Produtos para o Consumidor/legislação & jurisprudência , Queimaduras Oculares/epidemiologia , Lasers/efeitos adversos , Transtornos da Visão/epidemiologia , Criança , Queimaduras Oculares/prevenção & controle , Humanos , Jogos e Brinquedos , Formulação de Políticas , Comportamento Problema , Comportamento Autodestrutivo , Reino Unido/epidemiologia , Transtornos da Visão/prevenção & controleRESUMO
Preeclampsia can be lethal to both mother and baby. The prominent symptoms of this syndrome are hypertension, proteinuria and oedema, resulting from an exaggerated aseptic systemic inflammatory response, triggered by placental factors shed into the maternal circulation. Syncytiotrophoblast microparticles (STBM) are one possible factor, shed when the placenta is exposed to stressors such as hypoxia/reperfusion. These can disrupt mitochondria, triggering apoptosis and necrosis, placental pathologies which are increased in preeclampsia. We tested the effects of antioxidant vitamins C (50 microM) and E (50 microM) on trophoblast in culture, using term villous cytotrophoblast preparations. Following Percoll gradient centrifugation and MHC class I expressing cell depletion of placenta digests, syncytial fragments were removed using anti-placental alkaline phosphatase antibody. This yielded cytotrophoblasts of consistently high purity. EGF (10 ng/ml) stimulated syncytialisation and hCG and progesterone production. However, mitochondrial induced apoptosis (MIA) was evident 96h post-isolation, as mitochondrial membrane potential loss and caspase 9 and caspase 3 activation. ROCK-1 cleavage and syncytiotrophoblast particle shedding increased concurrently with apoptosis induction. Vitamins blocked MIA and syncytiotrophoblast particle shedding and significantly increased hCG (p<0.005) and progesterone (p<0.02) concentrations in culture supernatants, reflecting the increased survival rates. Although more cells survived in culture, syncytialisation rate (%) was significantly reduced (p<0.005). We conclude that vitamins C and E can significantly reduce mitochondrial damage generated following syncytialisation in vitro. However, further work is required to determine whether antioxidant vitamins interfere with normal fusion processes.
Assuntos
Ácido Ascórbico/farmacologia , Placenta/efeitos dos fármacos , Nascimento a Termo , Trofoblastos/efeitos dos fármacos , Vitamina E/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Fusão Celular , Células Cultivadas , Feminino , Hormônios/metabolismo , Humanos , Placenta/fisiologia , Placenta/ultraestrutura , Gravidez , Nascimento a Termo/fisiologia , Trofoblastos/metabolismo , Trofoblastos/fisiologia , Trofoblastos/ultraestruturaRESUMO
Lipid rafts are detergent-insoluble, low-density membrane domains that are rich in cholesterol and sphingolipids; caveolae are a subdomain of the biochemically defined glycolipid raft whose expression is associated with the protein caveolin-1. This protein associates with numerous signalling molecules, regulating their activity by holding them inactive. Human villous cytotrophoblasts contain caveolin-1, but levels reduce greatly during their differentiation into syncytiotrophoblast. Since caveolin-1 is a known regulator of apoptosis and trophoblast syncytialisation involves the apoptotic cascade, we hypothesised that cytotrophoblast caveolin-1 may also play a role in regulating fusion events involved in syncytium formation. The BeWo choriocarcinoma cell line has previously proved valuable for studying trophoblast syncytialisation, hence the present work was carried out to determine whether BeWo cells could be used as a model for the exploration of caveolin-1's role in regulating the syncytialisation process. Undifferentiated BeWo cells were found to express caveolin-1 in similar amounts to villous cytotrophoblasts isolated from term placenta. Lipid raft fractions prepared from these BeWo cells at confluence contained the raft-associated proteins caveolin-1 and -2, flotillin-1 and -2, stomatin and the heterotrimeric G protein, Galphaq. Confocal immunofluorescence studies revealed that caveolin-1 is internalized to the mitochondria, but not to the Golgi or endoplasmic reticulum, in subconfluent BeWo and that the protein relocates to the plasma membrane upon confluence, an observation confirmed by caveolin-1 and cytochrome c Western blotting of lipid raft fractions and mitochondria purified from confluent and subconfluent cells. Western blotting and immunofluorescence experiments comparing undifferentiated cells and those induced to differentiate using the cAMP analogue, dibutyryl cAMP, showed that BeWo syncytialisation was accompanied by a reduction in caveolin-1 levels, similar to the situation in primary villous cytotrophoblasts. Confluent, undifferentiated BeWo cultures were then used to investigate the cellular localisation of Rock-1, a protein which promotes cytoskeletal re-organisation important for syncytialisation and apoptosis. Its association with caveolin-1 was evidenced by the demonstration that the 160kDa proenzyme form of Rock-1 co-immunoprecipitates with caveolin-1 and vice versa, as well as by the co-localisation of the two proteins at the plasma membrane, as shown in immunofluorescence studies. A proportion of the total cell Rock-1 content was found in BeWo lipid raft fractions, confirming its membrane presence in confluent cells. This close association of plasmalemmal caveolin-1 with Rock-1 protein raises the possibility that caveolin-1 may regulate Rock-1 in these trophoblasts. We conclude that cell-cell contact is required for BeWo trophoblast to exhibit plasmalemmal caveolin-1; BeWo cells at confluence offer a useful model for the study of trophoblast raft behaviour during syncytialisation and for the exploration of the potential Rock-1-regulating role of caveolin-1 in this process.
Assuntos
Caveolina 1/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Trofoblastos/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Imunofluorescência , Expressão Gênica , Humanos , Imunoprecipitação , Microscopia Confocal , Mitocôndrias/metabolismo , Quinases Associadas a rhoRESUMO
CONTEXT: Placental urocortin has a role in the cascade of events leading to parturition by stimulating myometrial contractility and placental uterotonins secretion. OBJECTIVE: The objective of this study was to evaluate urocortin levels in maternal and fetal [umbilical cord artery (UCA) and vein (UCV)] plasma at term and preterm labor. DESIGN: The study design was a controlled cross-sectional study performed from November 2003 to June 2004. SETTING: This study was performed at the Division of Obstetrics and Gynecology, University of Siena (Siena, Italy). PATIENTS: Plasma samples were collected at term in the absence of labor (TNL; n = 27; 39.3 +/- 0.1 gestational weeks), at term spontaneous vaginal delivery (TL; n = 24; 40.1 +/- 0.2 gestational weeks), and at preterm labor (PTL; n = 19; 32.4 +/- 0.4 gestational weeks). Changes in urocortin mRNA expression were also evaluated in placentas collected from TNL (n = 11), TL (n = 11), and PTL (n = 10). INTERVENTION: Urocortin levels were measured by specific RIA. Changes in placental mRNA expression were determined by real-time quantitative RT-PCR analysis. RESULTS: Maternal and UCA plasma urocortin levels were significantly (P < 0.0001 for all) higher in TL and PTL than in TNL. Furthermore, UCA concentrations were significantly (P < 0.0001 for all) higher than and correlated with maternal concentrations (TNL: r = 0.45; P < 0.05; TL: r = 0.959; P < 0.0001; PTL: r = 0.7719; P < 0.0001). UCV levels were significantly (P < 0.001) higher in TL and PTL than in TNL and were significantly (P < 0.0001 for all) higher than and significantly (P < 0.0001 for all) correlated with maternal values, but were significantly (P < 0.0001 for all) lower than and correlated with UCA values (TNL: r = 0.9548; P < 0.0001; TL: r = 0.927; P < 0.0001; PTL: r = 0.838; P < 0.0001). Placental urocortin mRNA expression did not differ among TNL, TL, and PTL samples. CONCLUSIONS: Fetal urocortin secretion is increased in term and preterm labor. Whether these changes are a consequence rather than a cause of human parturition remains to be addressed.
Assuntos
Hormônio Liberador da Corticotropina/sangue , Sangue Fetal , Trabalho de Parto/sangue , Trabalho de Parto Prematuro/sangue , Hormônio Liberador da Corticotropina/genética , Estudos Transversais , Feminino , Idade Gestacional , Humanos , Concentração Osmolar , Placenta/metabolismo , Gravidez , RNA Mensageiro/metabolismo , Cordão Umbilical , Veias Umbilicais , UrocortinasRESUMO
Acute injection (sc) of the antiglucocorticoid RU-486 (5-10 mg/kg) stimulated oxygen consumption (VO2) and brown adipose tissue (BAT) activity (mitochondrial GDP binding) in the rat. A peak effect was seen 60-80 min after injection. The rise in VO2 was prevented by prior injection of the beta-adrenergic antagonist propranolol, and the effect on BAT was abolished by surgical denervation of the sympathetic supply to the tissue. Central injection (cerebroventricular) of a much lower dose (3-8 micrograms/kg) of RU-486 also stimulated VO2, and this effect was inhibited by a CRF receptor antagonist [alpha-helical CRF-(9-41)]. Peripheral injection of RU-486 also elicited acute thermogenic responses in older (greater than 12 months) rats and in genetically obese (Zucker) rats. In lean animals, daily injection of RU-486 inhibited weight gain and stimulated BAT without affecting food intake. The thermogenic effects of RU-486 appear to be due to central stimulation of sympathetic outflow and may involve CRF release. The data support previous studies on the effects of adrenalectomy and CRF on thermogenesis in the rat.
Assuntos
Tecido Adiposo/metabolismo , Temperatura Corporal , Hormônio Liberador da Corticotropina/fisiologia , Estrenos/farmacologia , Glucocorticoides/antagonistas & inibidores , Consumo de Oxigênio/efeitos dos fármacos , Sistema Nervoso Simpático/fisiologia , Animais , Masculino , Mifepristona , Ratos , Ratos EndogâmicosRESUMO
Stalk median eminence (SME) extracts were preincubated with antibodies to ovine corticotropin-releasing factor (oCRF) and/or vasopressin, and the resulting CRF bioactivity tested with the isolated anterior pituitary cell column bioassay. The ACTH-releasing ability of Wistar rat SME was reduced by 60% with vasopressin antiserum, by 53% with oCRF antiserum, and by 81% after incubation with both antisera simultaneously. SME-stimulated LH release was unaffected by these antisera, which were all used at a dilution of 1:1000. The ACTH-releasing activity of SME could not be completely abolished by increasing the oCRF antibody concentration, or, in the case of ovine SME, by decreasing the tissue concentration preincubated with oCRF antibodies. With Brattleboro SME (which contains no endogenous vasopressin) ACTH-releasing activity was reduced by 37%, 51%, and 57% with anti-oCRF at dilutions of 1:5000, 1:1000, and 1:500, respectively, but could not be reduced further by more concentrated antisera. We conclude, therefore, that the CRF bioactivity of rat SME is probably not due solely to an oCRF-like peptide, but that other substances, one being vasopressin, contribute to its ACTH-releasing ability.
Assuntos
Hormônio Liberador da Corticotropina/fisiologia , Hipotálamo/análise , Vasopressinas/fisiologia , Animais , Anticorpos/imunologia , Hormônio Liberador da Corticotropina/imunologia , Feminino , Adeno-Hipófise/análise , Ratos , Ratos Brattleboro , Ratos Endogâmicos , Ovinos , Vasopressinas/imunologiaRESUMO
Intact handled rats were pretreated with the immunoglobulin G fractions from normal rabbit serum or antisera to ovine corticotropin-releasing factor (CRF) and/or vasopressin and subjected to restraint or formalin stress. The formalin-induced rise in plasma ACTH was reduced to 28% in rats pretreated with anti-CRF, to 53% in those pretreated with antivasopressin, and to 16% in rats given both antibodies. Pretreatment of animals with anti-CRF, antivasopressin, or a combination of both antibodies also attenuated the ACTH response to restraint stress to 13%, 37%, and 12%, respectively, of those in normal rabbit serum-treated rats. Antiserum pretreatment did not reduce the restraint- or formalin-induced rise in plasma PRL in the same animals, however. We conclude, therefore, that both vasopressin and an ovine CRF-like peptide are physiologically relevant peptides involved in stress-induced ACTH release.
Assuntos
Hormônio Adrenocorticotrópico/antagonistas & inibidores , Hormônio Liberador da Corticotropina/imunologia , Soros Imunes/farmacologia , Estresse Fisiológico/metabolismo , Vasopressinas/imunologia , Hormônio Adrenocorticotrópico/metabolismo , Animais , Água Corporal/metabolismo , Formaldeído/farmacologia , Masculino , Ratos , Ratos Endogâmicos , Restrição Física , Ovinos , Estresse Fisiológico/induzido quimicamenteRESUMO
This study is the first to use CRH-binding protein (CRH-BP) purified from human plasma to investigate how the CRH-BP affects the physiological activity of CRH. After incubation at 37 C for 15 min, purified CRH-BP reduced the ACTH-releasing activity of synthetic human (h) CRH in a dose-dependent fashion; using hCRH and CRH-BP at concentrations commonly found in late gestational maternal plasma, (1.5 and 100 ng/mL, respectively) an average 76% reduction in ACTH release was obtained. No effect was observed under the same conditions on ACTH release induced by ovine (o) CRH, which does not bind to CRH-BP. Kinetic estimations are presented to show that the extent of binding of CRH to its BP (and, hence, the reduction in its bioactivity) depends on the time available for binding and the concentration of reactants. Equilibrium between CRH and its BP at the concentrations used in the bioactivity studies take place within 400 s. Since long term secretion of placental CRH into the peripheral circulation occurs during the third trimester of pregnancy, we suggest that the presence of circulating CRH BP may partly explain how markedly elevated plasma levels of CRH coexist with normal ACTH levels at this time. We also propose that stress-induced CRH release will not be similarly quenched by the CRH-BP in the hypothalamic portal system, as the concentration of CRH released will be high, and the exposure time before reaching pituitary corticotropes will be low. Using pituitary cells constantly bathed in BP premixed with hCRH or BP alone (to mimic the situations in pregnancy and nonpregnancy, respectively), we show that short concentrated pulses of synthetic CRH (10 ng in 5 s) or rat stalk median eminence extract (one tenth stalk median eminence in 5 s) retain their ability to induce ACTH secretion despite the presence of CRH-BP. It is, thus, possible that CRH can still exert its role as a stress hormone in late gestation.
Assuntos
Hormônio Adrenocorticotrópico/biossíntese , Hormônio Liberador da Corticotropina/metabolismo , Hipotálamo/metabolismo , Placenta/metabolismo , Receptores de Neurotransmissores/metabolismo , Animais , Bovinos , Cromatografia em Gel , Relação Dose-Resposta a Droga , Feminino , Humanos , Técnicas In Vitro , Eminência Mediana/fisiologia , Hipófise/metabolismo , Gravidez , Ratos , Receptores de Hormônio Liberador da CorticotropinaRESUMO
A "two-site" immunoradiometric assay (IRMA) which allows the direct estimation of human CRH (hCRH) in plasma is described. Using this IRMA, basal levels of CRH in normal subjects ranged from 2-28 pg/mL [mean, 15 +/- 7 (+/- SD) pg/mL; n = 58]. Values in men and women were similar. Plasma CRH values within this range were also found in patients with Cushing's syndrome, Addison's disease, and Nelson's syndrome, with no correlation between plasma CRH and ACTH levels in these patients. Elevated plasma CRH levels were found in pregnant women near term [1462 +/- 752 (+/- SD) pg/mL; n = 55], and the dilution curve of this CRH-like immunoreactivity paralleled the IRMA standard curve. After its immunoadsorption from maternal plasma, this CRH-like material eluted on reverse phase high performance liquid chromatography with a retention time identical to that of synthetic CRH and had equipotent bioactivity with the synthetic peptide in the perfused anterior pituitary cell bioassay. Circulating CRH was not detected in Wistar rats, even after adrenalectomy and subsequent ether stress. Synthetic hCRH was degraded by fresh human plasma relatively slowly; 65% of added CRH remained after 1 h of incubation at 37 C. Degradation was inhibited by heat treatment (54 C; 1 h), cold treatment (4 C; 4 h), or freezing and thawing. Loss of synthetic rat CRH occurred more rapidly when fresh rat plasma was used; only 20% of added CRH remained under the same conditions. The inability to measure CRH in peripheral rat plasma may be due to the presence of active CRH-degrading enzymes which fragment the CRH molecule into forms not recognized by the CRH IRMA.
Assuntos
Hormônio Liberador da Corticotropina/sangue , Doença de Addison/sangue , Adrenalectomia , Animais , Bioensaio , Cromatografia Líquida de Alta Pressão , Hormônio Liberador da Corticotropina/farmacologia , Síndrome de Cushing/sangue , Feminino , Humanos , Imunoensaio , Radioisótopos do Iodo , Masculino , Síndrome de Nelson/sangue , Adeno-Hipófise/efeitos dos fármacos , Gravidez , Ratos , Valores de Referência , Estresse Fisiológico/sangueRESUMO
Plasma CRH was measured in maternal plasma throughout the third trimester of pregnancy, during labor, and postpartum. CRH levels were also measured in arterial and venous umbilical cord plasma samples. In normal pregnant women, plasma CRH increased from 50 +/- 15 (+/- SEM) pg/mL at 28 weeks gestation (n = 41) to 1462 +/- 182 pg/mL at 40 weeks (n = 55) and 1680 +/- 101 pg/mL (n = 65) in labor. Women with pregnancy-induced hypertension (n = 49) had plasma CRH levels significantly elevated above this normal range. Similarly, women who subsequently went into premature labor had raised levels several weeks before the onset of labor. After delivery, plasma CRH returned to normal within 15 h. Total plasma cortisol levels varied little throughout the third trimester, but increased during labor and remained elevated 2-3 days postpartum. There was, therefore, no correlation between plasma cortisol and CRH, implying that this placental CRH is not primarily involved in the control of the maternal hypothalamo-pituitary adrenal axis during pregnancy. The concentrations of CRH in umbilical cord plasma samples were considerably lower than those in the maternal circulation and were close to those in normal nonpregnant adults.
Assuntos
Hormônio Liberador da Corticotropina/sangue , Trabalho de Parto/sangue , Gravidez/sangue , Adolescente , Adulto , Feminino , Sangue Fetal/metabolismo , Humanos , Hipertensão/sangue , Trabalho de Parto Prematuro/sangue , Complicações Cardiovasculares na Gravidez/sangue , Artérias Umbilicais , Veias UmbilicaisRESUMO
This study examined the different molecular forms of CRH in normal and preeclampsia maternal plasma and protease-blocked placental extracts using antibodies to different regions of the CRH precursor, pro-CRH. In the absence of protease inhibitors, chromatographed normal placental extracts contained four peaks of immunoreactivity corresponding to unprocessed approximately 19-kDa pro-CRH, its approximately 8-kDa intermediate metabolite, pro-CRH125-194, its approximately 2.8-kDa midportion fragment, pro-CRH125-151, and 4.75-kDa CRH1-41. However, if protease inhibitors were included in the extraction medium, only pro-CRH and pro-CRH125-194 were found. Pro-CRH processing was more extensive in protease-blocked preeclampsia placentas than in those from normal pregnancy, with three peaks corresponding to pro-CRH, proCRH125-194, and mature CRH1-41 peptide found. Using quantitative competitive PCR, the messenger ribonucleic acid levels of CRH precursor in preeclampsia placentas were 1.7-fold higher than those in normal placentas (37.83 +/- 3.48 vs. 21.83 +/- 2.59 attomoles/microg total ribonucleic acid, respectively; P < 0.005). Preeclampsia placentas contained significantly more CRH1-41 cross-reactivity (4.72 +/- 1.22 pmol/g) than normal term placentas (1.52 +/- 0.39 pmol/g; P < 0.048) extracted in medium containing protease inhibitors. The content of pro-CRH(125+/-151)-reactive species in these extracts followed the same pattern, with more immunoreactivity detected in preeclampsia placentas (4.23 +/- 1.39 pmol/g) than in those from normal term pregnancies (1.44 +/- 0.32 pmol/g; P < 0.01). Sequential plasma samples from 10 women with normal pregnancy and 5 women with preeclampsia were assayed for pro-CRH(125-151)- and CRH(1-41)-immunoreactive species In normal pregnancy, maternal plasma CRH(1-41) immunoreactivity rose with increasing gestational age, reaching 460 +/- 48 pmol/L at term. In women with preeclampsia, CRH(1-41) levels at each gestational age point were higher than those at the equivalent stage of normal pregnancy. In contrast, the levels of pro-CRH(125-151)-immunoreactive species remained barely detectable throughout normal and preeclamptic pregnancy. Both pro-CRH and CRH(1-41), but not pro-CRH(125-151), were shown to bind to the plasma CRH-binding protein. Our findings highlight the importance of protection of placental tissue from degrading enzymes during extraction and show that most of the CRH in the human placenta exists as unprocessed pro-CRH, with very little in the form of CRH(1-41) except in preeclampsia. Our studies using maternal plasma indicate that CRH(1-41) is the only one of the pro-CRH fragments studied to be maintained in significant amounts in the maternal circulation and also the only fragment studied for which a specific plasma binding protein exists.
Assuntos
Hormônio Liberador da Corticotropina/metabolismo , Pré-Eclâmpsia/metabolismo , Gravidez/metabolismo , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Células CHO , Hormônio Liberador da Corticotropina/sangue , Hormônio Liberador da Corticotropina/química , Hormônio Liberador da Corticotropina/genética , Cricetinae , Feminino , Humanos , Fragmentos de Peptídeos/sangue , Fragmentos de Peptídeos/metabolismo , Placenta/metabolismo , Gravidez/sangue , Precursores de Proteínas/sangue , Precursores de Proteínas/química , Precursores de Proteínas/genética , RNA Mensageiro/metabolismo , Valores de ReferênciaRESUMO
In pregnancy, maternal plasma corticotropin releasing hormone (CRH) concentrations rise substantially in the third trimester and fall rapidly post-partum. A binding protein (BP) specific for CRH exists in the human circulation which inactivates CRH, thus possibly explaining why maternal ACTH does not rise outside normal limits throughout gestation. We here describe the measurement of CRH-BP directly in plasma during human pregnancy using a radioimmunoassay that is not affected by the presence of the high plasma levels of CRH that occur at this time. In 119 healthy non-pregnant individuals, mean CRH-BP levels were 4.46 nmol/L +/- 1.0 (SD), with a wide range of 1.81-7.24 nmol/L. Plasma CRH-BP in 34 pregnant women randomly sampled during the first and second trimesters also averaged 4.46 nmol/L +/- 1.54, with individual values ranging from 1.59-7.51 nmol/L and there was no correlation of CRH-BP levels with gestational age. In a group of 14 women sampled sequentially throughout the third trimester, plasma CRH-BP averaged 4.56 nmol/L +/- 1.70 at 30-35 weeks gestation and fell dramatically to 1.84 nmol/L +/- 0.43 at weeks 38-40 (P < 0.001). The post partum recovery in CRH-BP levels occurred within 48 hours of delivery. These results indicate that there is an increase in the availability of free, potentially bioactive CRH at term to stimulate the release of ACTH from the maternal pituitary and/or to act at a peripheral, non-pituitary CRH receptor(s).
Assuntos
Proteínas de Transporte/sangue , Hormônio Liberador da Corticotropina/sangue , Gravidez/sangue , Adulto , Proteínas de Transporte/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Cinética , Pessoa de Meia-Idade , Peso Molecular , Período Pós-Parto/sangue , Segundo Trimestre da Gravidez , Terceiro Trimestre da Gravidez , Proteínas Recombinantes/sangue , Proteínas Recombinantes/isolamento & purificação , Valores de ReferênciaRESUMO
OBJECTIVE: Two clinical trials investigated the pharmacokinetics of human corticotropin-releasing factor (hCRF), resulting cortisol release, and associated hemodynamic changes. METHODS: In a 3 x 3 Latin square design, subjects were randomized to receive a single dose of 5 microg x kg(-1) hCRF as a 10-minute intravenous infusion, a 180-minute infusion, and a subcutaneous injection in separate study sessions 7 days apart. Twelve additional subjects obtained a subcutaneous dose of either 300, 600, or 1200 microg hCRF on 3 consecutive days. Noncompartmental and compartmental pharmacokinetic analysis was performed. Hemodynamic response was characterized with use of pharmacodynamic models. RESULTS: The volume of distribution at steady state was 9.81 +/- 3.0 and 15.61 +/- 2.9, and the clearance was 256 +/- 40 mL x min(-1) and 345 +/- 90 mL x min(-1) for the 10-minute and 180-minute intravenous infusion, respectively (P < .05). Corresponding elimination half-life was 45 +/- 7 minutes and 37 +/- 10 minutes. Two-compartment and 1-compartment models adequately described the 10-minute and 180-minute infusions, respectively. The bioavailability of hCRF after subcutaneous administration was 67% +/- 17%. Apparent clearance remained unchanged for different subcutaneous doses. Peak plasma cortisol concentrations were similar after subcutaneous and intravenous administration of hCRF. Repetitive administration of hCRF did not result in accumulation but produced a reduced plasma cortisol response. A sigmoidal model related plasma hCRF concentrations to increase in heart rate (maximum, 39 beats x min(-1)). The relationship between the modest decrease in diastolic blood pressure and plasma hCRF concentrations was linear. CONCLUSION: The pharmacokinetics of intravenously administered hCRF were nonlinear, but apparent clearance was constant for various subcutaneous doses. An excellent bioavailability and preserved bioactivity make the subcutaneous route an attractive choice. Repetitive administration of hCRF probably caused tolerance of the cortisol response.
Assuntos
Hormônio Liberador da Corticotropina/administração & dosagem , Hormônio Liberador da Corticotropina/farmacocinética , Hemodinâmica/efeitos dos fármacos , Hidrocortisona/sangue , Adulto , Análise de Variância , Disponibilidade Biológica , Feminino , Meia-Vida , Humanos , Infusões Intravenosas , Injeções Subcutâneas , MasculinoRESUMO
It is suggested that corticotrophin-releasing hormone (CRH) is involved in parturition. We have previously reported the presence of the CRH receptor subtype 1 (CRH R1) in human uterine myocytes. The aim of the present study was to investigate whether expression of the CRH R1 in myometrial tissue changes in pregnancy and labour. We used a quantitative competitive PCR method to measure the mRNA levels of this receptor in non-pregnant and in term pregnant myometrium before and at different stages of labour. The levels of mRNA for the housekeeping gene for glucocerebrosidase (GCB) were also determined. The results were expressed as a ratio of CRH R1 and GCB mRNA levels. We have found that in pregnancy the CRH R1 is down-regulated from a ratio of 0.093+/-0.011 in non-pregnant myometrium to 0.012+/-0.005 (P<0.001) in term non-labouring myometrium. No significant changes were observed in the CRH R1:GCB ratio in tissues sampled within 13 h (0.013+/-0.004) from the start of labour. In summary, normalised levels of CRH R1 are down-regulated in pregnancy and do not change during labour. We speculate that our results do not support a direct role for the CRH R1 receptor in myometrial stimulation.
Assuntos
Trabalho de Parto , Miométrio/metabolismo , Gravidez , RNA Mensageiro/metabolismo , Receptores de Hormônio Liberador da Corticotropina/genética , Adulto , Animais , Feminino , Humanos , Camundongos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
We have expressed human pre-procorticotrophin-releasing hormone (pre-proCRH) as a fusion protein to beta-galactosidase in Escherichia coli. The chimeric fusion protein was found in insoluble bacterial inclusion bodies. The inclusion bodies were isolated, purified and solubilized, and used as imunogens in rabbits to raise antibodies against the neuropeptide moiety. The antibodies generated were characterized by immunoassays and immunocytochemical techniques. The immunoassay results showed that the recombinant pre-proCRH antibodies cross-reacted with the full-length CRH precursor and several cleavage products derived from it, i.e. CRH(1-41) and CRH(36-41). They did not cross-react with the CRH antagonist CRH(9-41). Extracts of stalk median eminence from various species were also studied. The antibodies cross-reacted with extracts from ovine, bovine, human and rat tissues, exhibiting parallel displacement curves to that of synthetic rat/human CRH(1-41) used as standard. They also cross-reacted with a skin extract of the frog, a species known to contain a CRH-related peptide, i.e. sauvagine, in this tissue. The immunocytochemical studies demonstrated that the antibodies generated against recombinant human pre-proCRH labelled neurones in the rat paraventricular nucleus of the hypothalamus. They exhibited the same pattern of staining as that obtained with an antibody generated against synthetic CRH(1-41). The results indicate that these antibodies can recognize CRH(1-41) or CRH-related molecules in the hypothalamus in situ as well as in tissue extracts from several species. Hence, they will be useful tools in the study of the CRH biosynthetic pathway and its intracellular compartmentalization.
Assuntos
Formação de Anticorpos , Hormônio Liberador da Corticotropina/biossíntese , Escherichia coli/imunologia , Haptenos/genética , Precursores de Proteínas/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , beta-Galactosidase/biossíntese , Animais , Bovinos , Hormônio Liberador da Corticotropina/imunologia , Reações Cruzadas , Escherichia coli/genética , Feminino , Haptenos/imunologia , Humanos , Hipotálamo/química , Técnicas Imunoenzimáticas , Masculino , Hipófise/química , Precursores de Proteínas/imunologia , Ratos , Ratos Endogâmicos , Proteínas Recombinantes de Fusão/imunologia , Ovinos , beta-Galactosidase/imunologiaRESUMO
Forty-one-residue corticotropin-releasing factor is a physiologically significant mediator of the hypothalamic control of corticotropin secretion by the anterior pituitary gland. This releasing hormone is produced by parvicellular neurons in the hypothalamic paraventricular nucleus that project to the external zone of the median eminence. Recent immunocytochemical evidence based on work with a rabbit antiserum against rat corticotropin-releasing factor (code rC70) suggests that about half of the parvicellular corticotropin-releasing factor-containing neurons in the hypothalamic paraventricular nucleus synthesize vasopressin, another potent corticotropin secretagogue, while the rest of the cells do not. If this is indeed the case, the neurohumoral control of corticotropin release may be mediated via distinct hypothalamic effector pathways utilizing releasing hormone cocktails of varying composition. In the present study we have examined the specificity of various antisera against rat corticotropin-releasing factor in immunocytochemical staining. Male Wistar rats pretreated with colchicine were used throughout. The brain was fixed by perfusion with a Zamboni type fixative solution. Vibratome sections of the hypothalamus were immunostained with three different primary antisera (codes rC70, rCRF-3, oCRF-N) using the peroxidase-antiperoxidase or avidin-biotin complex methods. All three antisera stained cell groups previously described to be immunopositive for corticotropin-releasing factor. Most notably, however, rC70 labelled a significant number of additional cells, most readily identified in the arcuate and suprachiasmatic nuclei, as well as in the dorsolateral hypothalamic area caudal to the paraventricular nucleus.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Especificidade de Anticorpos , Hormônio Liberador da Corticotropina/imunologia , Reações Cruzadas , Hipotálamo/metabolismo , Soros Imunes/imunologia , Animais , Hormônio Liberador da Corticotropina/metabolismo , Hipotálamo/citologia , Imuno-Histoquímica , Masculino , Ratos , Ratos EndogâmicosRESUMO
We investigated the intracellular localization of CRH in transiently transfected COS-7 cells expressing the full-length rat corticotropin-releasing hormone (CRH) precursor cDNA. CRH synthesized by transfected COS-7 cells is mainly stored intracellularly. In contrast, CHO-K1 cells expressing the same CRH precursor stored and released equal amounts of immunoreactive (IR)-CRH. Ultrastructural analysis revealed that CRH is stored in electron-dense aggregates in the RER of transiently transfected COS-7 cells and does not migrate into the Golgi apparatus. On the basis of the different intracellular localization, storage, and release of CRH in COS-7 and CHO-K1 cells, we hypothesize that the intracellular trafficking of CRH within the constitutive secretory pathway for protein secretion not only depends on its primary amino acid sequence but might also be influenced by intracellular conditions or factors.
Assuntos
Células COS/metabolismo , Hormônio Liberador da Corticotropina/metabolismo , Animais , Células CHO , Células COS/ultraestrutura , Cricetinae , Retículo Endoplasmático Rugoso/metabolismo , Complexo de Golgi/metabolismo , Microscopia Eletrônica , Ratos , TransfecçãoRESUMO
The reported effects of corticotrophin-releasing hormone (CRH) on human myometrium support the existence of specific receptors for the hormone in this tissue. We have used the reverse transcriptase-polymerase chain reaction (RT-PCR) technique to study the expression of mRNA coding for the CRH R1 and R2 receptors. RT-PCR of total RNA from both nonpregnant and pregnant myometrium using specific primers resulted in amplification products of the expected sizes for the R1 alpha and R2 alpha CRH receptors. The identity of these amplification products was confirmed by specific restriction digests and sequencing. Immunohistochemistry using a rabbit antibody raised against a specific domain of the CRH R1 receptor demonstrated that the R1 mRNA is translated into protein and confirmed that it is the uterine smooth muscle cells from both nonpregnant and pregnant women that bear this receptor. Our results suggest that CRH may play a role in human pregnancy at the myometrium.