RESUMO
BACKGROUND: Diagnosis of pheochromocytoma (PC) is based on a combination of clinical suspicion, finding an adrenal mass, increased plasma, and urine concentrations of catecholamine metabolites and is finally confirmed with histopathology. In human medicine, it is controversial whether biochemically testing plasma is superior to testing urine. OBJECTIVES: To measure urinary and plasma catecholamines and metanephrines in healthy dogs, dogs with PC, hypercortisolism (HC), and nonadrenal diseases (NAD) and to determine the test with the best diagnostic performance for dogs with PC. ANIMALS: Seven PC dogs, 10 dogs with HC, 14 dogs with NAD, 10 healthy dogs. METHODS: Prospective diagnostic clinical study. Urine and heparin plasma samples were collected and stored at -80°C before analysis using high-pressure liquid chromatography (HPLC) coupled to electrochemical detection or tandem mass spectrometry were performed. Urinary variables were expressed as ratios to urinary creatinine concentration. RESULTS: Dogs with PC had significantly higher urinary normetanephrine and metanephrine:creatinine ratios and significantly higher plasma-total and free normetanephrine and plasma-free metanephrine concentrations compared to the 3 other groups. There were no overlapping results of urinary normetanephrine concentrations between PC and all other groups, and only one PC dog with a plasma normetanephrine concentration in the range of the dogs with HC and NAD disease. Performances of total and free plasma variables were similar. Overlap of epinephrine and norepinephrine results between the groups was large with both urine and plasma. CONCLUSION AND CLINICAL IMPORTANCE: Measurement of normetanephrine is the preferred biochemical test for PC and urine was superior to plasma.
Assuntos
Neoplasias das Glândulas Suprarrenais/veterinária , Catecolaminas/urina , Síndrome de Cushing/veterinária , Doenças do Cão/urina , Normetanefrina/urina , Feocromocitoma/veterinária , Neoplasias das Glândulas Suprarrenais/sangue , Neoplasias das Glândulas Suprarrenais/urina , Animais , Catecolaminas/sangue , Síndrome de Cushing/sangue , Síndrome de Cushing/urina , Doenças do Cão/sangue , Cães , Feminino , Masculino , Normetanefrina/sangue , Feocromocitoma/sangue , Feocromocitoma/urinaRESUMO
BACKGROUND: Decreased dipeptidylpeptidase IV (DPPIV) activity within the human nasal mucosa has previously been shown to contribute to the severity of chronic inflammatory rhinosinusitis. OBJECTIVE: To investigate and correlate the role of DPPIV activity with regard to bronchial inflammation. METHODS: DPPIV/CD26 activity/concentration was investigated in the bronchial tissue of human subjects suffering from chronic bronchial inflammation. In addition, the effect of a recombinant Aspergillus fumigatus DPPIV (fuDPPIV) was investigated on histamine-induced bronchoconstriction in anesthetized rabbits. RESULTS AND CONCLUSIONS: DPPIV/CD26 was present in submucosal seromucous glands, in leukocytes and to a very low degree in endothelial cells of human bronchi. DPPIV activity was correlated with tissue CD26 content measured by immunoassay. As previously reported for the nasal mucosa, DPPIV/CD26 activity was inversely correlated with the degree of airway inflammation. Systemic pretreatment with recombinant fuDPPIV markedly reduced the increase in histamine-induced airway resistance in rabbits. In conclusion, DPPIV activity modulates lower airway tone by degrading unknown peptidic substrates released by histamine in response to an allergen. Contrasting with our observations in the nose, this modulation is apparently not mediated via a neurokinin (NK1) receptor.
Assuntos
Hiper-Reatividade Brônquica/enzimologia , Bronquite Crônica/enzimologia , Dipeptidil Peptidase 4/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Animais , Biomarcadores/metabolismo , Hiper-Reatividade Brônquica/prevenção & controle , Bronquite Crônica/patologia , Broncoconstrição/efeitos dos fármacos , Dipeptidil Peptidase 4/farmacologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Histamina/farmacologia , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Mucosa Nasal/enzimologia , Mucosa Nasal/fisiopatologia , Probabilidade , Coelhos , Valores de Referência , Estudos de Amostragem , Sensibilidade e Especificidade , Índice de Gravidade de Doença , Substância P/farmacologiaRESUMO
HYPOTHESIS: Neuropeptides released from sensory nerves may contribute to airway inflammation, particularly if their metabolism is impaired through defective inactivation and/or increased production. In the airways, neuropeptides are subjected to degradation by enzymes such as dipeptidyl peptidase (DPP-IV), and are upregulated by neurotrophins such as brain derived neurotrophic factor (BDNF). We therefore assessed in primary human nasal epithelial cells the expression of DPP-IV and BDNF under basal and inflammatory conditions. METHODS: Human epithelial cells were isolated from nasal polyps and middle turbinates, and grown on collagen-coated polycarbonate filters with an air liquid-interface. After three weeks of culture, constitutive cellular expression of DPP-IV and BDNF was assessed by measuring its activity and by ELISA, respectively. To mimick in vivo inflammatory conditions, cells were exposed apically and basolaterally to 50 ng/ml of TNFalpha, IL-1beta, and IFN-gamma for two days. DPP-IV activity and BDNF protein expression were measured in cell lysates and in the apical and basolateral media. RESULTS: Constitutive DPP-IV activity was similar in polyps and turbinates cells. In contrast, polyps epithelial cells expressed higher amounts of BDNF compared to turbinates derived cells. On the other hand, TNFalpha, IL-1beta, and IFN-gamma did not affect DPP-IV activity but significantly increased the cellular expression and the basolateral secretion of BDNF. CONCLUSIONS: Our data show for the first time that primary human airway epithelial cells produced DPP-IV and BDNF under basal conditions. Furthermore, the production and secretion of BDNF were markedly increased in response to pro-inflammatory cytokines, confirming the involvement of BDNF in airway inflammation.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/biossíntese , Dipeptidil Peptidase 4/biossíntese , Células Epiteliais/metabolismo , Cavidade Nasal/citologia , Pólipos Nasais/metabolismo , Fator Neurotrófico Derivado do Encéfalo/análise , Células Cultivadas , Dipeptidil Peptidase 4/análise , Células Epiteliais/química , Humanos , Pólipos Nasais/química , Conchas NasaisRESUMO
The aim of the present work was to find out whether NPY synthesized in human adrenal chromaffin cells controls in an autocrine/paracrine fashion the release of catecholamines by these cells. Accordingly, the constitutive and regulated release of both NPY and catecholamines was measured simultaneously in cultured human chromaffin cells. In addition, by using both RT-PCR and a combination of specific agonists and antagonists, we characterized the expression of NPY receptors on these cells as well as their pharmacology. Our results were as follows. 1) Human chromaffin cells constitutively secrete NPY. 2) Nicotine elicits a rapid increase in the release of both catecholamines and NPY; this release of NPY is more sustained than that of catecholamines. 3) RT-PCR shows expression of Y1, Y2, Y4, and Y5 receptor mRNA by chromaffin cells; these receptors are functional, as various receptor specific agonists elicit an increase in intracellular calcium. 4) Peptide YY, in contrast to NPY, is not able to stimulate the release of catecholamines. This finding was corroborated by the observation that no receptor-specific antagonists were able to reduce constitutive catecholamine release, whereas an NPY-immunoneutralizing antibody markedly attenuated the secretion. Taken together, these data suggest that NPY originating from the adrenal medulla locally enhances the secretion of catecholamines, presumably by acting via the putative y3 receptor.
Assuntos
Glândulas Suprarrenais/metabolismo , Catecolaminas/metabolismo , Células Cromafins/metabolismo , Neuropeptídeo Y/fisiologia , Adolescente , Glândulas Suprarrenais/citologia , Glândulas Suprarrenais/efeitos dos fármacos , Adulto , Células Cultivadas , Criança , Células Cromafins/efeitos dos fármacos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neuropeptídeo Y/análogos & derivados , Neuropeptídeo Y/metabolismo , Neuropeptídeo Y/farmacologia , Nicotina/farmacologia , Peptídeo YY/farmacologia , Receptores de Neuropeptídeo Y/metabolismo , Receptores de Neuropeptídeo Y/fisiologia , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: Plasma free metanephrines are a more reliable analyte to measure than catecholamines for the biochemical diagnosis of pheochromocytomas. We hypothesized that the long persistence of total (sulfate-conjugated plus free) metanephrines in the blood might have a significant diagnostic value. METHODS: We measured plasma concentrations of catecholamines and total metanephrines (sulfate-conjugated plus free forms) by HPLC with amperometric detection, and neuropeptide Y (NPY) by an amplified ELISA in seven patients before and after removal of their pheochromocytomas. The results for catecholamine, total metanephrines, and NPY in each patient were analyzed for up to 120 min, starting from the time of tumor vessel clamping. The persistence of analytes was quantified as the area under the concentration-time curve over 120 min. RESULTS: On the basis of the upper reference limit for each variable, plasma free norepinephrine (NE) and epinephrine (E) concentrations were increased preoperatively in at least one sample in seven and six patients, respectively. Total normetanephrine (NMN) and metanephrine (MN) were increased in all samples in seven and six patients, respectively. NPY was increased 2- to 465-fold. After removal of the tumor, MN and NMN showed a higher average relative increase above the upper limit of the reference interval than NE and E (P = 0.05), whereas NPY was intermediate. The persistence of increased values was significantly shorter for catecholamines than for metanephrines. The half-life estimated by nonlinear regression was 12.3 +/- 7.8 min for NPY. Significant correlations were observed among NE, E, NMN, MN, and NPY concentrations, but parent markers (E and MN or NE and NMN) did not appear significantly intercorrelated. CONCLUSIONS: A larger increase and a longer persistence of total metanephrines (reflecting predominantly sulfo-conjugated metanephrines) than catecholamines and NPY in plasma may contribute to their greater diagnostic accuracy in pheochromocytoma.
Assuntos
Catecolaminas/sangue , Metanefrina/sangue , Neuropeptídeo Y/sangue , Feocromocitoma/sangue , Feminino , Meia-Vida , Humanos , Masculino , Feocromocitoma/cirurgiaRESUMO
Neuropeptide Y (NPY) regulates neurotransmitter release through activation of the Y2 receptor subtype. We have recently characterized a human glioblastoma cell line, LN319, that expresses exclusively NPY Y2 receptors and have demonstrated that NPY triggers transient decreases in cAMP and increases in intracellular calcium responses. The present study was designed to further characterize calcium signalling by NPY and bradykinin (BK) in LN319 cells. Both agonists elevated free intracellular calcium ([Ca(2+)](i)) without soliciting calcium influx. NPY appeared to activate two distinct signalling cascades that liberate calcium from thapsigargin- and ryanodine-insensitive compartments. One pathway proceeded through phospholipase C (PLC)-dependent phosphatidylinositol turnover, while the other triggered calcium release through a so far unidentified mediator. Part of the response was sensitive to pertussis toxin (PTX) under conditions where the toxin totally abolished the NPY-mediated effects on cAMP. The calcium release induced by BK on the other hand was largely PTX-insensitive, PLC-dependent, and from both thapsigargin- and ryanodine-sensitive stores. Following stimulation with NPY, subsequent [Ca(2+)](i) responses to NPY were strongly depressed. Partial heterologous desensitization occurred, when BK was used as the first agonist, whereas NPY had no effect on a subsequent stimulation with BK. These data suggest that NPY-induced calcium mobilization in LN319 cells involves two different G proteins and signalling mediators, and a hitherto unidentified calcium compartment. Homologous desensitization of NPY signalling might be explained by receptor-G protein uncoupling, while heterologous desensitization by BK could be the result of either transient depletion or inhibition of a mediator in the calcium signalling cascades activated by NPY.
Assuntos
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Receptores de Neuropeptídeo Y/química , Receptores de Neuropeptídeo Y/metabolismo , Animais , Bradicinina/farmacologia , Cálcio/farmacologia , AMP Cíclico/metabolismo , Estrenos/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Humanos , Fosfatos de Inositol/metabolismo , Neuropeptídeo Y/farmacologia , Toxina Pertussis , Inibidores de Fosfodiesterase/farmacologia , Ligação Proteica , Pirrolidinonas/farmacologia , Rianodina/metabolismo , Transdução de Sinais , Suínos , Tapsigargina/metabolismo , Tapsigargina/farmacologia , Células Tumorais Cultivadas , Fosfolipases Tipo C/metabolismo , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Propofol is a widely used anesthetic for both induction and maintenance of anesthesia during surgery. A strong feeling of hunger has been reported during the early recovery period after propofol anesthesia. We have investigated the effect of propofol on appetite in 10 patients undergoing a craniotomy and in parallel measured neuropeptide Y (NPY), catecholamines, and serotonin levels in the cerebrospinal fluid and plasma during anesthesia. Ten patients anesthetized with a volatile agent (isoflurane) served as a control group. Plasma NPY and catecholamines levels were not affected by surgery at any time. We observed a strong increase in NPY concentrations in the cerebrospinal fluid independently of the anesthetic technique agent used, whereas catecholamines were unchanged. We found that serotonin concentrations decreased significantly in the plasma (but not in the cerebrospinal fluid) of patients treated by propofol when compared with the control group; this decrease was associated with an increase of hunger early postoperatively. We concluded that the proappetite effect of propofol is mediated through a decrease of serotonin at the peripheral level.
Assuntos
Anestésicos/farmacologia , Apetite/efeitos dos fármacos , Catecolaminas/sangue , Isoflurano/farmacologia , Neuropeptídeo Y/sangue , Propofol/farmacologia , Serotonina/sangue , Adulto , Idoso , Catecolaminas/líquido cefalorraquidiano , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neuropeptídeo Y/líquido cefalorraquidiano , Serotonina/líquido cefalorraquidianoRESUMO
Neuropeptide Y (NPY) gene is expressed in human pituitary gland where its function is partially elucidated. NPY could act as a neuroendocrine modulator within this gland. This study was undertaken to assess whether NPY expression is correlated to various pathological situations. Using a highly specific anti-NPY monoclonal antibody, immunohistochemistry analysis was performed in surgically removed pituitary glands. The study included biopsies from 112 human pituitary adenomas, 12 hyperplastic glands and normal anterior pituitary tissues in 34 cases. NPY is immunodetected in 33% of all adenomas, 25% hyperplastic glands and 12% of non-tumoral pituitary gland. NPY expression was significantly higher in adenomas compared to the normal gland. However, no correlation was observed between NPY content and the type of hormonal secretion, sex, age and the status of tumour proliferating potential.
Assuntos
Neuropeptídeo Y/metabolismo , Neoplasias Hipofisárias/metabolismo , Adenoma/metabolismo , Adenoma/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais , Criança , Pré-Escolar , Feminino , Gonadotropinas Hipofisárias/metabolismo , Hormônio do Crescimento Humano/metabolismo , Humanos , Hiperplasia , Imuno-Histoquímica , Lactente , Masculino , Pessoa de Meia-Idade , Neuropeptídeo Y/imunologia , Adeno-Hipófise/metabolismo , Adeno-Hipófise/patologia , Neoplasias Hipofisárias/patologiaRESUMO
Sympathetic nerves have long been suspected of trophic activity, but the nature of their angiogenic factor has not been determined. Neuropeptide Y (NPY), a sympathetic cotransmitter, is the most abundant peptide in the heart and the brain. It is released during nerve activation and ischemia and causes vasoconstriction and smooth muscle cell proliferation. Here we report the first evidence that NPY is angiogenic. At low physiological concentrations, in vitro, it promotes vessel sprouting and adhesion, migration, proliferation, and capillary tube formation by human endothelial cells. In vivo, in a murine angiogenic assay, NPY is angiogenic and is as potent as a basic fibroblast growth factor. The NPY action is specific and is mediated by Y1 and Y2 receptors. The expression of both receptors is upregulated during cell growth; however, Y2 appears to be the main NPY angiogenic receptor. Its upregulation parallels the NPY-induced capillary tube formation on reconstituted basement membrane (Matrigel); the Y2 agonist mimics the tube-forming activity of NPY, whereas the Y2 antagonist blocks it. Endothelium contains not only NPY receptors but also peptide itself, its mRNA, and the "NPY-converting enzyme" dipeptidyl peptidase IV (both protein and mRNA), which terminates the Y1 activity of NPY and cleaves the Tyr1-Pro2 from NPY to form an angiogenic Y2 agonist, NPY3-36. Endothelium is thus not only the site of action of NPY but also the origin of the autocrine NPY system, which, together with the sympathetic nerves, may be important in angiogenesis during tissue development and repair.
Assuntos
Endotélio Vascular/química , Neovascularização Fisiológica/efeitos dos fármacos , Neuropeptídeo Y/fisiologia , Sistema Nervoso Simpático/química , Animais , Aorta/efeitos dos fármacos , Capilares , Colágeno/farmacologia , Dipeptidil Peptidase 4/biossíntese , Dipeptidil Peptidase 4/genética , Dipeptidil Peptidase 4/fisiologia , Combinação de Medicamentos , Fatores de Crescimento Endotelial/farmacologia , Feminino , Fator 2 de Crescimento de Fibroblastos/farmacologia , Expressão Gênica , Humanos , Laminina/farmacologia , Linfocinas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neuropeptídeo Y/biossíntese , Neuropeptídeo Y/genética , Neuropeptídeo Y/isolamento & purificação , Neuropeptídeo Y/metabolismo , Neuropeptídeo Y/farmacologia , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Reação em Cadeia da Polimerase , Proteoglicanas/farmacologia , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Receptores de Neuropeptídeo Y/biossíntese , Receptores de Neuropeptídeo Y/efeitos dos fármacos , Receptores de Neuropeptídeo Y/genética , Receptores de Neuropeptídeo Y/fisiologia , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Neuropeptide Y (NPY) is a 36-amino-acid neurotransmitter which is widely distributed throughout the central and peripheral nervous system. NPY involvement has been suggested in various physiological responses including cardiovascular homeostasis and the hypothalamic control of food intake. At least six subtypes of NPY receptors have been described. Because of the lack of selective antagonists, the specific role of each receptor subtype has been difficult to establish. Here we describe mice deficient for the expression of the Y1 receptor subtype. Homozygous mutant mice demonstrate a complete absence of blood pressure response to NPY, whereas they retain normal response to other vasoconstrictors. Daily food intake, as well as NPY-stimulated feeding, are only slightly diminished, whereas fast-induced refeeding is markedly reduced. Adult mice lacking the NPY Y1 receptor are characterized by increased body fat with no change in protein content. The higher energetic efficiency of mutant mice might result, in part, from the lower metabolic rate measured during the active period, associated with reduced locomotor activity. These results demonstrate the importance of NPY Y1 receptors in NPY-mediated cardiovascular response and in the regulation of body weight through central control of energy expenditure. In addition, these data are also indicative of a role for the Y1 receptor in the control of food intake.
Assuntos
Fenômenos Fisiológicos Cardiovasculares , Sistema Cardiovascular/fisiopatologia , Comportamento Alimentar/fisiologia , Atividade Motora/fisiologia , Receptores de Neuropeptídeo Y/deficiência , Animais , Sistema Cardiovascular/metabolismo , Feminino , Expressão Gênica/genética , Genótipo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Atividade Motora/genética , Mutagênese Sítio-Dirigida , Mutação/genética , Neuropeptídeo Y/sangue , Neuropeptídeo Y/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/genética , Receptores de Neuropeptídeo Y/genética , Receptores de Neuropeptídeo Y/fisiologiaRESUMO
Neuropeptide Y (NPY) is a potent inhibitor of neurotransmitter release through the Y2 receptor subtype. Specific antagonists for the Y2 receptors have not yet been described. Based on the concept of template-assembled synthetic proteins we have used a cyclic template molecule containing two beta-turn mimetics for covalent attachment of four COOH-terminal fragments RQRYNH2 (NPY 33-36), termed T4-[NPY(33-36)]4. This structurally defined template-assembled synthetic protein has been tested for binding using SK-N-MC and LN319 cell lines that express the Y1 and Y2 receptor, respectively. T4-[NPY(33-36)]4 binds to the Y2 receptor with high affinity (IC50 = 67.2 nM) and has poor binding to the Y1 receptor. This peptidomimetic tested on LN319 cells at concentrations up to 10 microM shows no inhibitory effect on forskolin-stimulated cAMP levels (IC50 for NPY = 2.5 nM). Furthermore, we used confocal microscopy to examine the NPY-induced increase in intracellular calcium in single LN319 cells. Preincubation of the cells with T4-[NPY(33-36)]4 shifted to the right the dose-response curves for intracellular mobilization of calcium induced by NPY at concentrations ranging from 0.1 nM to 10 microM. Finally, we assessed the competitive antagonistic properties of T4-[NPY(33-36)]4 at presynaptic peptidergic Y2 receptors modulating noradrenaline release. the compound T4-[NPY(33-36)]4 caused a marked shift to the right of the concentration-response curve of NPY 13-36, a Y2-selective fragment, yielding a pA2 value of 8.48. Thus, to our best knowledge, T4-[NPY(33-36)]4 represents the first potent and selective Y2 antagonist.
Assuntos
Neuropeptídeo Y/antagonistas & inibidores , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/farmacologia , Receptores de Neuropeptídeo Y/antagonistas & inibidores , Ligação Competitiva , Cálcio/metabolismo , AMP Cíclico/metabolismo , Humanos , Norepinefrina/metabolismo , Peptídeos Cíclicos/metabolismo , Receptores de Neuropeptídeo Y/metabolismo , Células Tumorais CultivadasRESUMO
Proneuropeptide Y (ProNPY) undergoes cleavage at a single dibasic site Lys38-Arg39 resulting in the formation of 1-39 amino acid NPY which is further processed successively by carboxypeptidase-like and peptidylglycine alpha-amidating monooxygenase enzymes. To investigate whether prohormone convertases are involved in ProNPY processing, a vaccinia virus derived expression system was used to coexpress recombinant ProNPY with each of the prohormone convertases PC1/3, PC2, furin, and PACE4 in Neuro2A and NIH 3T3 cell lines as regulated neuroendocrine and constitutive prototype cell lines, respectively. The analysis of processed products shows that only PC1/3 generates NPY in NIH 3T3 cells while both PC1/3 and PC2 are able to generate NPY in Neuro2A cells. The convertases furin and PACE4 are unable to process ProNPY in either cell line. Moreover, comparative in vitro cleavage of recombinant NPY precursor by the enzymes PC1/3, PC2 and furin shows that only PC1/3 and PC2 are involved in specific cleavage of the dibasic site. Kinetic studies demonstrate that PC1/3 cleaves ProNPY more efficiently than PC2. The main difference between the cleavage efficiency is observed in the Vmax values whereas no major difference is observed in Km values. In addition the cleavage by PC1/3 and PC2 of two peptides reproducing the dibasic cleavage site with different amino acid sequence lengths namely (20-49)-ProNPY and (28-43)-ProNPY was studied. These shortened ProNPY substrates, when recognized by the enzymes, are more efficiently cleaved than ProNPY itself. The shortest peptide is not cleaved by PC2 while it is by PC1/3. On the basis of these observations it is proposed, first, that the constitutive secreted NPY does not result from the cleavage carried out by ubiquitously expressed enzymes furin and PACE4; second, that PC1/3 and PC2 are not equipotent in the cleavage of ProNPY; and third, substrate peptide length might discriminate PC1/3 and PC2 processing activity.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Neuropeptídeo Y/metabolismo , Pró-Proteína Convertase 1 , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Serina Endopeptidases/metabolismo , Subtilisinas/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Ácido Aspártico Endopeptidases/genética , Furina , Expressão Gênica , Vetores Genéticos , Cinética , Espectrometria de Massas , Camundongos , Dados de Sequência Molecular , Neuropeptídeo Y/genética , Fragmentos de Peptídeos/análise , Pró-Proteína Convertase 2 , Pró-Proteína Convertases , Precursores de Proteínas/genética , Ratos , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/genética , Especificidade por Substrato , Subtilisinas/genética , Células Tumorais Cultivadas , Vaccinia virus/genéticaRESUMO
Neuropeptide Y (NPY) is a 36 aminoacid peptide known to inhibit glucose-stimulated insulin secretion. NPY has been shown to be synthesized and secreted by rat islets of Langerhans. More recently, we described the presence on NPY within human islets of Langerhans and in several pancreatic endocrine tumors. In this report, we describe the case of a patient presenting with an insulinoma who underwent the surgical resection of the tumor and was studied in vivo and in vitro for NPY production. Using a highly specific and sensitive two-site amplified enzyme-linked immunosorbent assay, we detected high plasma NPY levels in the patient prior to the surgical resection of the tumor which returned to normal after surgery. NPY was secreted from the tumor when kept in primary cell culture. Furthermore, immunohistochemistry of the insulinoma revealed the presence of NPY and its C-flanking peptide together with insulin, chromogranin and neuron specific enolase. It is concluded that elevated circulating NPY levels observed in this patient with an insulinoma reflected in vivo secretion by the tumor and it is hypothesized that NPY could potentially be used as an endocrine marker in patients with suspected insulinoma.
Assuntos
Insulinoma/metabolismo , Neuropeptídeo Y/metabolismo , Neoplasias Pancreáticas/metabolismo , Idoso , Idoso de 80 Anos ou mais , Cromograninas/análise , Feminino , Humanos , Técnicas Imunoenzimáticas , Imuno-Histoquímica , Insulina/análise , Insulinoma/química , Insulinoma/cirurgia , Neuropeptídeo Y/análise , Neuropeptídeo Y/sangue , Neoplasias Pancreáticas/química , Neoplasias Pancreáticas/cirurgia , Fosfopiruvato Hidratase/análise , Células Tumorais CultivadasRESUMO
OBJECTIVE: The observation that phaeochromocytoma possess specific somatostatin binding sites led us to test the hypothesis that octreotide may have antisecretory potential in patients with phaeochromocytoma. We therefore compared the effects of octreotide and placebo on blood pressure and plasma catecholamines and neuropeptide Y. PATIENTS: Ten consecutive patients referred to a tertiary care centre for the diagnosis and treatment of a phaeochromocytoma. DESIGN AND MEASUREMENTS: We performed a crossover comparison of either three 100 micrograms subcutaneous injections of octreotide over one day or 3 injections of octreotide vehicle over another. Blood pressure was measured over 24 hours on each test day using an automatic ambulatory recorder. Blood samples were collected before (at 0800 and 0900 h) and after (at 1000, 1100, 1200, 1300 and 1500 h) placebo or octreotide injection. Plasma catecholamines were assayed by high-performance liquid chromatography and neuropeptide Y was determined using a two-site amplified enzyme immunoassay. All patients then underwent surgery and tumoral somatostatin binding site density was determined by quantitative autoradiography. RESULTS: Compared to placebo, octreotide did not alter mean 24-hour ambulatory blood pressure or plasma neuropeptide Y, or plasma or urinary catecholamine, levels. Although a moderate reduction in plasma noradrenaline was found in the two patients with the highest tumoral somatostatin binding site densities, overall octreotide-induced variations in plasma noradrenaline did not correlate with somatostatin binding site density. Blood glucose increased from 5.4 +/- 0.3 on placebo to 7.8 +/- 0.5 mmol/l on octreotide (P < 0.01). CONCLUSION: In the present controlled conditions, short-term administration of octreotide had no antisecretory effect in patients with phaeochromocytoma.
Assuntos
Neoplasias das Glândulas Suprarrenais/tratamento farmacológico , Pressão Sanguínea/efeitos dos fármacos , Catecolaminas/sangue , Neuropeptídeo Y/sangue , Octreotida/uso terapêutico , Feocromocitoma/tratamento farmacológico , Neoplasias das Glândulas Suprarrenais/metabolismo , Adulto , Monitorização Ambulatorial da Pressão Arterial , Estudos Cross-Over , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Feocromocitoma/metabolismo , Receptores de Somatostatina/metabolismo , Método Simples-CegoRESUMO
Neuropeptide Y (NPY) is a 36 amino acid peptide known to inhibit glucose-stimulated insulin secretion. NPY has recently been shown to be synthetized within rat islets of Langerhans and to be secreted in a differentiated rat insulin-secreting cell line, and as to this date the localization of NPY in human endocrine pancreas has not been reported. As NPY shares high amino acid sequence homology with peptide YY (PYY) and pancreatic polypeptide (PP), the polyclonal antibodies raised against these peptides often cross-react with each other. To demonstrate the presence of NPY in the human endocrine pancreas, we used a highly specific monoclonal antibody raised against NPY and another against its C-flanking peptide (CPON). We studied three cases of hyperplasia of Langerhans islets and 11 cases of endocrine tumors of the pancreas. NPY and CPON were detected in all three cases of hyperplasia. For the 11 pancreatic tumors, five and nine of the tumors were positive for the antibodies NPY and CPON, respectively. The two negative tumors for CPON immunoreactivity were differentiated insulinomas, which showed no evidence of other hormonal secretion. In normal Langerhans islet, NPY and CPON immunoreactivities were colocalized in glucagon-producing cells (alpha-cells) and in a few insulin-secreting cell (beta-cells).(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Ilhotas Pancreáticas/patologia , Neuropeptídeo Y/análise , Hormônios Pancreáticos/análise , Neoplasias Pancreáticas/patologia , Adulto , Idoso , Animais , Anticorpos , Tumor Carcinoide/patologia , Carcinoma/patologia , Linhagem Celular , Feminino , Humanos , Hiperplasia , Imuno-Histoquímica/métodos , Insulina/análise , Insulinoma/patologia , Ilhotas Pancreáticas/citologia , Masculino , Pessoa de Meia-Idade , Tumores Neuroendócrinos/patologia , RatosRESUMO
The present data show that monoclonal antibodies (NPY02, NPY03, NPY04, NPY05) directed against 4 distinct epitopes on NPY may have different actions on NPY binding and NPY-induced cellular responses. NPY02 and NPY05 recognize the 11-24 and 32-36 amidated form of NPY, respectively. These 2 antibodies block the binding of NPY to its receptor as well as the NPY-induced inhibition of cAMP accumulation caused in SK-N-MC cells by forskolin. NPY02 and NPY05 have also an inhibitory action on NPY-induced contraction of rabbit femoral arteries. NPY03 and NPY04 are directed against the 27-34 and 1-12 part of NPY, respectively. NPY03 and NPY04 inhibit the binding of NPY only at very high concentrations and have a weak effect on cAMP response to NPY. NPY02 and NPY05 might provide useful tools to study the effect of NPY in cellular systems and organ preparations.
Assuntos
Neuropeptídeo Y/metabolismo , Receptores de Neuropeptídeo Y/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais , AMP Cíclico/metabolismo , Mapeamento de Epitopos , Humanos , Técnicas Imunológicas , Técnicas In Vitro , Dados de Sequência Molecular , Ensaio RadioliganteRESUMO
The potential role of angiotensin-II in mediating catecholamine and neuropeptide-Y release in a human pheochromocytoma has been investigated. Angiotensin-II type I receptors are transcribed and translated into functional proteins in a surgically removed pheochromocytoma. Primary cell culture of the tumor has been studied in a perfused system. Angiotensin-II increased the release of norepinephrine and neuropeptide-Y by the pheochromocytes. Activation of the angiotensin-II type I receptors by angiotensin-II was associated with a rise in cytosolic free calcium. The renin-angiotensin system may, therefore, contribute to the secretion of catecholamines and NPY occurring in patients with pheochromocytoma and when stimulated trigger hypertensive crisis.
Assuntos
Neoplasias das Glândulas Suprarrenais/metabolismo , Angiotensina II/farmacologia , Neuropeptídeo Y/metabolismo , Norepinefrina/metabolismo , Feocromocitoma/metabolismo , Adulto , Cálcio/metabolismo , Expressão Gênica , Humanos , Masculino , Neuropeptídeo Y/genética , Perfusão , Receptores de Angiotensina/efeitos dos fármacos , Receptores de Angiotensina/genética , Receptores de Angiotensina/fisiologia , Sistema Renina-Angiotensina/fisiologia , Células Tumorais CultivadasRESUMO
The ability to express exogenous mammalian genes stably in post-mitotic cells such as neurons remains an important goal for those attempting to modulate neurotransmission through gene delivery. We therefore investigated how differentiation to a post-mitotic state affected the expression of an exogenous gene encoding for neuropeptide Y (NPY) following transfection with an adeno-associated virus (AAV) derived vector. This vector (pJDT95npy) was constructed with rat NPY cDNA (551 bp) inserted downstream from the indigenous AAV p5, p19 and p40 promoters to characterize their relative abilities to drive NPY mRNA expression. Transfection of dividing neuroblastoma CHP126 cells with pJDT95npy resulted in the differential expression of chimeric NPY mRNAs derived from each promoter. P40-driven species became dominant after 1 month post-transfection. Vector integration into chromosomal DNA was demonstrated by Southern blot analyses, indicating at least some region-selective integration. In dividing cell extracts, only a low level of pro-NPY immunoreactivity and no mature NPY immunoreactivity was recovered. However, after differentiation of the pJDT95npy-transfected CHP 126 cells to a post-mitotic state, significant levels of pro-NPY and mature NPY were recovered in the cells and media. Differentiation also had a time-dependent effect on mRNA expression: a spike of p5 driven expression on day 3 was followed predominantly by p40-driven expression on day 5. This study indicates that AAV-derived vectors using the p40 promoter may be used to express genes in post-mitotic cells such as neurons.
Assuntos
Dependovirus/genética , Expressão Gênica , Neuroblastoma/metabolismo , Neuropeptídeo Y/biossíntese , Animais , Northern Blotting , Southern Blotting , Bucladesina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Divisão Celular , Linhagem Celular , Vetores Genéticos , Humanos , Mitose , Plasmídeos , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Ratos , Proteínas Recombinantes/biossíntese , Mapeamento por Restrição , Transfecção , Células Tumorais CultivadasRESUMO
We produced and characterized three anti-C-flanking peptides of neuropeptide Y (CPON) monoclonal antibodies. The Ka for these antibodies ranged from 0.4 to 0.8 x 10(8) l/mol with an IC50 for CPON(1-30) at about 20 nM as determined by ELISA. All these antibodies are IgG1 and recognize the 16-30 part of CPON. These antibodies and a specific anti-NPY monoclonal antibody were used to study the localization of CPON and NPY in the human kidney. The avidin-biotin technique was employed. NPY and CPON immunoreactivities were present in large amount in the renal tubules of the human kidney but not in the glomeruli. No labeling was found within the renal arterioles and veins, but some immunoreactivity was evidenced in the perivascular area. Because no specific receptor for CPON has been described to date, the presence of this peptide in the tubules may be due to a tubular reabsorption or perhaps to a local synthesis of pro-NPY.