RESUMO
The purpose of this report is to analyse the long-term outcome in hydrocephalic children treated by shunt insertion, in particular their psycho-intellectual development and quality of life. We studied 46 patients aged 3 - 21, who had been operated on in the Department of Paediatric Surgery of the Medical University of Bialystok between 1982 - 2000 and had had ventriculo-peritoneal shunts inserted during their first year of life. Data from anamnesis and medical documentation were analysed. Age-appropriate psychomotor development and IQ tests were carried out: Wechsler Intelligence Scale for Children, Revised (WISC-R) (for children between the ages of 6 and 16), Wechsler Adult Intelligence Scale, Revised (WAIS-R PL) (for adults), Brunet-Lezine psychometric scale (early childhood), and Terman-Merril intelligence scale (children younger than 3). The final IQs were above 90 in 30 % of children, between 70 and 90 in 24 %, between 50 and 70 in 26 %, and lower than 50 in 20 %. 69 % of patients presented with neurological deficits and visual or auditory deficits were found in 22 %. Integration into normal schools was possible for 58.7 % of the children, one of whom is now a second year medical student. A relationship between shunt malfunction and the children's development was observed. An essential aspect of caring for hydrocephalic children is their rehabilitation and integration into society. Early physical rehabilitation, stimulation of psychological development, and continued monitoring by a paediatric surgeon to ensure proper functioning of the shunts will improve the independence of such children in their families and among their peers.
Assuntos
Hidrocefalia/cirurgia , Qualidade de Vida , Derivação Ventriculoperitoneal , Adolescente , Adulto , Criança , Pré-Escolar , Educação Inclusiva , Humanos , Hidrocefalia/reabilitação , Masculino , Desempenho PsicomotorRESUMO
OBJECTIVE: The analysis occurrence of developmental defects of newborns in the City Hospital in Bydgoszcz during the period 1995-1999. MATERIALS AND METHODS: Over the period of 5 years, 14,943 newborns delivered in City Hospital in Bydgoszcz. The group of 358 (2.40%) with developmental anomalies was analysed retrospectively. The subject of the study was to estimate the types of malformations and their frequency. RESULTS: In our material cardiovascular anomalies (29.05%), reproductive system anomalies (20.11%) and muscosceletal anomalies (18.72%) were most frequent. Breech-presented or transverse lie fetuses had double the risk of any anomalies. In addition, pre-term delivery were not positively associated with any newborn defects. The frequency of occurrence cesarean sections was positively correlated with presence of congenital malformations.
Assuntos
Anormalidades Congênitas/epidemiologia , Área Programática de Saúde , Hospitais Urbanos , Humanos , Recém-Nascido , Polônia/epidemiologia , Estudos RetrospectivosRESUMO
Complement receptor type 1 is expressed by erythrocytes and most leukocytes. A soluble form is shed from the leukocytes and found in plasma (sCR1). sCR1 is a powerful inhibitor of complement. We report an increased sCR1 in the plasma of leukemia patients, up to levels producing measurable complement inhibition. Half of the 180 patients with acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL) had sCR1 levels above the normal range. The highest levels were observed in T-ALL (17 patients). The complement function of a T-ALL serum was improved by blocking sCR1 with a specific mAb (3D9). Measurements in 16 peripheral stein cell donors before and after granulocyte colony-stimulating factor (G-CSF) administration showed an increase in sCR1 (before, 43.8+/-15.4; at day 5, 118.3+/-44.7 ng/mL; P < 0.0001). This increase paralleled the increase in total leukocyte counts and was concomitant with de novo leukocyte mRNA CR1 expression in all three individuals tested. Whether pharmacological intervention may be used to up-regulate sCR1 so as to inhibit complement in vivo should be further investigated.
Assuntos
Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Leucemia Linfoide/sangue , Leucemia Linfoide/tratamento farmacológico , Leucemia Mieloide/sangue , Leucemia Mieloide/tratamento farmacológico , Receptores de Complemento 3b/sangue , Animais , Proteínas do Sistema Complemento/fisiologia , Ensaio de Imunoadsorção Enzimática , Transplante de Células-Tronco Hematopoéticas , Humanos , Selectina L/sangue , Coelhos , Estudos Retrospectivos , Solubilidade , Doadores de TecidosRESUMO
We describe a simple, effective approach to the creation of autologous tissue-engineered cartilage in the shape of a human nipple by injecting a reverse thermosensitive polymer seeded with autologous chondrocytes in an immunocompetent porcine animal model. A biodegradable, biocompatible copolymer of polyethylene oxide and polypropylene oxide (Pluronic F-127), which exists as a liquid below 4 degrees C and polymerizes to a thick gel when it is exposed to physiologic temperatures (body temperatures), was used as a vehicle for chondrocyte delivery and as a scaffold to guide growth. Autologous chondrocytes isolated from porcine auricular elastic cartilage and suspended in 30% (weight/volume) Pluronic F-127 were injected on the ventral surface of the pigs from which the cells had been isolated. A circumferential subdermal suture was used to support the contour of the implant and assist in its projection in the form of a human nipple. After 3 weeks, the skin over and surrounding the implant was tattooed to create the appearance of a human nipple-areolar complex. As controls, an equal number of injections were made using either cells alone (not suspended in hydrogel), or hydrogel alone. After 10 weeks, all specimens were excised and examined both grossly and histologically. Before harvesting, visual inspection of the tattooed chondrocyte-Pluronic F-127 hydrogel implant sites revealed that they closely resembled a human female nipple-areolar complex. Nodules were similar in size, shape, and texture to a human nipple at each injection site. Glistening opalescent tissue was surgically isolated from each implant site. Hematoxylin and eosin, safranine o, trichrome blue, and Verhoeff's stains of the experimental implants showed nodules with the characteristic histologic signs of elastic cartilage. Control injections of copolymer hydrogel alone exhibited no evidence of cartilage formation. Control injections of chondrocytes alone showed evidence of dissociated microscopic nodules of elastic cartilage.
Assuntos
Materiais Biocompatíveis , Condrócitos/transplante , Mamilos/cirurgia , Procedimentos de Cirurgia Plástica/métodos , Poloxâmero/administração & dosagem , Polietilenoglicóis/administração & dosagem , Próteses e Implantes , Animais , Masculino , Suínos , TatuagemRESUMO
This study investigated the effect of the non-peptidic B2 bradykinin (BK) receptor antagonist WIN 64338 on BK binding and BK-induced inositol phosphate formation on guinea-pig tracheal smooth muscle cells in culture. The presence of specific and saturable binding sites for BK was demonstrated using [3H]BK. Scatchard analysis indicates a single population of binding sites for [3H]BK with a maximal density (Bmax) of 245.4 +/- 71 fmol/mg of protein and an equilibrium dissociation constant (Kd) of 87.7 +/- 0.12 pM. The order of potency of] B2 BK receptor ligands was Hoe 140 = NPC 17731 > BK > WIN 64338 > D- Arg0[Hyp3, D-Phe7]-BK > > des-Arg9-BK, while B1 BK receptor antagonist, des-Arg9-[Leu8]-BK, was without effect on [3H]BK binding. These results demonstrate the presence of B2 Bk receptors on cultured tracheal smooth muscle cells. The cells were stimulated by BK, and inositol phosphate formation was determined by anion exchange chromatography. The stimulating effect of BK on inositol phosphate formation was concentration dependent (1 nM to 10 microM). The B1 BK agonist des-Arg9-BK did not induce inositol phosphate formation. The order of potency of B2 antagonists to inhibit BK-induced inositol phosphate formation was Hoe 140 = NPC 17731 > WIN 64338 > D-Arg0[Hyp3, D-Phe7]-BK. This study demonstrates that WIN 64338 is able to displace [3H]BK binding and to inhibit B2-BK-induced inositol phosphate formation on cultured guinea-pig tracheal smooth muscle cells.
Assuntos
Antagonistas dos Receptores da Bradicinina , Músculo Liso/efeitos dos fármacos , Naftalenos/farmacologia , Compostos Organofosforados/farmacologia , Traqueia/efeitos dos fármacos , Antagonistas Adrenérgicos beta/farmacologia , Analgésicos/farmacologia , Animais , Ligação Competitiva , Bradicinina/análogos & derivados , Bradicinina/metabolismo , Bradicinina/farmacologia , Células Cultivadas/efeitos dos fármacos , Imunofluorescência , Cobaias , Fosfatos de Inositol/metabolismo , Masculino , Músculo Liso/citologia , Naftalenos/metabolismo , Oligopeptídeos/farmacologia , Compostos Organofosforados/metabolismo , Receptor B2 da Bradicinina , Receptores da Bradicinina/biossíntese , Receptores da Bradicinina/efeitos dos fármacosRESUMO
OBJECTIVE: To investigate synovial fluid (SF) for the presence of CR1 and to study its relationship to SF leukocytes and to serum levels of soluble CR1 (sCR1) in patients with rheumatic diseases. METHODS: Synovial fluids were collected from 35 patients with rheumatoid arthritis (RA) and 26 patients with other inflammatory joint diseases. Total CR1 in the SF and serum were measured with a sandwich enzyme-linked immunosorbent assay (ELISA) that recognized both soluble and transmembrane forms of CR1. The characteristics of CR1 in SF were analyzed by ultracentrifugation and by a second ELISA specific for transmembrane CR1. RESULTS: CR1 was found in all SF samples tested (range 5-281 ng/ml). SF CR1 was higher in patients with RA (mean +/- SD 81 +/- 66 ng/ml) than in those with other inflammatory joint diseases (31.8 +/- 23.8 ng/ml) (P < 0.001). Serum sCR1 was not significantly increased in the patients compared with the normal subjects. There was no correlation between serum sCR1 and SF CR1. In 44% of the patients, the SF CR1 level was higher than the serum sCR1 level. A fraction (30-80%) of SF CR1 was pelleted by ultracentrifugation and, unlike serum sCR1, it reacted in an ELISA specific for transmembrane CR1. Thus, SF contained 2 forms of CR1: a membrane-associated and a soluble form, which was confirmed by sucrose density-gradient ultracentrifugation. SF CR1 levels correlated directly with the number of SF total leukocytes and polymorphonuclear leukocytes (PMN). These 2 forms of CR1 were also found in the supernatant of in vitro-activated PMN from normal subjects. SF CR1 exhibited the capacity to act as a cofactor for the factor I degradation of C3b. CONCLUSION: CR1 is found in the SF of patients with joint inflammation. The data suggest that SF CR1 originates from the infiltrating leukocytes, which shed both a soluble and a membrane-associated form. Whether SF CR1 participates in the local regulation of complement activation remains to be examined.
Assuntos
Artrite/metabolismo , Receptores de Complemento/análise , Líquido Sinovial/química , Artrite/sangue , Artrite Reumatoide/sangue , Artrite Reumatoide/metabolismo , Centrifugação com Gradiente de Concentração/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Humanos , Immunoblotting , Neutrófilos/metabolismo , Osteoartrite/sangue , Osteoartrite/metabolismo , Receptores de Complemento/sangue , Dodecilsulfato de Sódio , Espondilite Anquilosante/sangue , Espondilite Anquilosante/metabolismo , Sacarose , UltracentrifugaçãoRESUMO
We investigated the effect of the in vivo treatment of guinea pigs with methylprednisolone, 10 mg/kg daily, on lung muscarinic and beta-adrenergic receptors. Receptor densities were assessed by saturation experiments of tritiated N-methylscopolamine and dihydroalprenolol binding to lung membranes. After 3 h of treatment, methylprednisolone induced a decrease of 19.2% (P < 0.05) of muscarinic receptors but was without effect on beta-adrenergic receptor density. After 24 h, an increase of 39.7% (P < 0.01) and 16.9% (P < 0.05) was observed for muscarinic and beta-adrenergic receptors, respectively. For muscarinic receptors, this increase reached 53.4% (P < 0.01) within 48 h and stayed at this level until 96 h. The increase of beta-adrenergic receptors was maximal (24.9%) after 72 h and returned to the control value after 96 h. The dissociation constant (Kd) values of both ligands were not affected by the glucocorticoid treatment. Functional studies showed that the 96 h treatment did not affect the contractile response of guinea pig lung parenchymal strips to carbachol since the 50% concentration value (EC50) and the maximal contraction value (Emax) were not significatively different from control values. These data show that glucocorticoids control the expression of both muscarinic and beta-adrenergic receptors in guinea pig lung but with different time courses and to a larger extent for muscarinic receptors. The glucocorticoid treatment did not modify the contractile response of lung strips to carbachol, confirming the absence of effect on the affinity of muscarinic receptors and suggesting that the receptor reserve exceed the increase of their density by the steroid.
Assuntos
Glucocorticoides/farmacologia , Pulmão/efeitos dos fármacos , Metilprednisolona/farmacologia , Receptores Adrenérgicos beta/efeitos dos fármacos , Receptores Muscarínicos/efeitos dos fármacos , Análise de Variância , Animais , Carbacol/farmacologia , Avaliação Pré-Clínica de Medicamentos , Cobaias , Técnicas In Vitro , Pulmão/metabolismo , Masculino , Membranas/efeitos dos fármacos , Membranas/metabolismo , Contração Muscular/efeitos dos fármacosRESUMO
The rat neuromedin B (NMB) receptor was expressed in Rat-1 fibroblasts to elucidate the signaling pathways and mitogenic effects mediated by this seven-transmembrane domain receptor. Receptor expression was verified by ligand binding and Ca2+ mobilization, which were blocked by the NMB receptor antagonist D-Nal-Cys-Tyr-D-Trp-Orn-Val-Cys-Nal-NH2. NMB acted as a potent growth factor promoting DNA synthesis and cell proliferation in serum-free medium in Rat-1 cells transfected with the NMB receptor. Prior to DNA synthesis, NMB stimulated phosphorylation of 80K/MARCKS, a major substrate of protein kinase C, which could be prevented by the selective protein kinase C inhibitor GF 109203X. Furthermore, NMB induced a rapid p42MAPK activation and tyrosine phosphorylation of multiple proteins including p125FAK and paxillin. The half-maximal concentrations (EC50) of NMB required to induce DNA synthesis (0.7-0.9 nM) and cell proliferation (0.7-1 nM) paralleled the Kd for 125I-[D-Tyr0]NMB binding and the EC50 values for the induction of the early signaling events. Thus, NMB can activate multiple signal transduction pathways and act as a sole mitogen through its receptor expressed in Rat-1 fibroblasts.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana , Mitógenos/farmacologia , Receptores da Bombesina/genética , Transdução de Sinais/fisiologia , Animais , Cálcio/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Moléculas de Adesão Celular/metabolismo , Divisão Celular/fisiologia , Linhagem Celular/enzimologia , Linhagem Celular/ultraestrutura , Proteínas do Citoesqueleto/metabolismo , DNA/biossíntese , Fibroblastos/enzimologia , Fibroblastos/ultraestrutura , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Substrato Quinase C Rico em Alanina Miristoilada , Paxilina , Fosfoproteínas/metabolismo , Fosforilação , Proteína Quinase C/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas/metabolismo , Ratos , Receptor de Insulina/metabolismo , Transfecção , Tirosina/metabolismoRESUMO
We have investigated the effects of a nitric oxide (NO) biosynthesis inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) on the bombesin-evoked contraction of guinea pig parenchymal lung strips. The bombesin-induced contractions of lung strips were significantly increased after L-NAME (300 micro)) pre-treatment. The maximal response was increased (P < 0.01) by 37% after L-NAME treatment when compared with the control group. The pD2 value was not influenced by L-NAME pre-treatment. The enhancement of the bombesin-induced contraction caused by L-NAME was reversed by addition of an excess of the NO precursor L-arginine (600 microM) but not by the addition of its inactive enantiomer D-arginine (600 microM). Like L-NAME, methylene blue (1 microM), an agent that inhibits the soluble guanylyl cyclase activated by NO, significantly increased (P < 0.01) the maximal contraction induced by bombesin (183 +/- 16 mg) when compared with the control group (141 +/- 15 mg). When tested against other agonist-induced contractions, L-NAME did not change the responsiveness of parenchymal lung strips to bradykinin or carbachol but significantly increased lung contraction induced by histamine. NO synthesis inhibition resulted in a pronounced increase in the bombesin-induced contraction of guinea-pig lung strips. Our results suggest that bombesin contributes to NO synthesis and release which then acts to reduce the contraction of the lungstrip in response to bombesin.
Assuntos
Bombesina/farmacologia , Pulmão/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Óxido Nítrico/fisiologia , Animais , Arginina/análogos & derivados , Arginina/farmacologia , Cobaias , Técnicas In Vitro , Pulmão/fisiologia , Masculino , NG-Nitroarginina Metil ÉsterRESUMO
Characterization of bombesin binding sites in healthy human lung was performed through direct binding techniques. There was limited binding in the absence of trypsin and chymotrypsin inhibitors, suggesting important activities of both enzymes in human lung and/or increased sensitivity of the bombesin sites toward them. In human lung membranes, bombesin, gastrin releasing peptide (GRP) and GRP-preferring bombesin receptor antagonists displaced [125I-Tyr4]bombesin binding with high affinities (36-177 nM), whereas neuromedin B possessed a lower affinity of 2878 nM. [D-F5Phe6,D-Ala11]bombesin-(6-13)-methyl ester, the most active GRP-preferring bombesin antagonist as yet reported, had the highest affinity among all antagonists tested whereas neuromedin B had the lowest affinity. These data demonstrate that the bombesin binding sites in the human lung are of the GRP-preferring type.
Assuntos
Bombesina/metabolismo , Pulmão/metabolismo , Peptídeos/metabolismo , Sítios de Ligação , Ligação Competitiva , Bombesina/análogos & derivados , Bombesina/antagonistas & inibidores , Carcinoma/metabolismo , Carcinoma/cirurgia , Peptídeo Liberador de Gastrina , Humanos , Pulmão/efeitos dos fármacos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/cirurgia , Neurocinina B/análogos & derivados , Neurocinina B/metabolismo , Fragmentos de Peptídeos/metabolismo , Peptídeos/farmacologiaRESUMO
The possible interaction of bombesin receptors with guanine nucleotide binding protein in guinea pig lung was studied. The non-hydrolysable GTP analogue guanosine-5'-[gamma-thio]triphosphate (GTP gamma S) was shown to decrease [125I-Tyr4]bombesin binding in a concentration-dependent manner. The specificity of this effect was assessed by examining the effects of other guanine nucleotides on this binding at a concentration of 1 mM. GMP and GDP weakly inhibited [125I-Tyr4]bombesin binding (2 and 19%, respectively), whereas GTP, guanosine-5'-[beta-thio]triphosphate (GDP beta S), and 5-guanylylimidodiphosphate (GppNHp) exhibited similar potencies, inducing 52%, 46%, and 43% inhibition of [125I-Tyr4]bombesin binding respectively. Saturation experiments performed in the absence and presence of 100 microM GTP gamma S indicated the presence of a single population of receptors in both cases. However, the addition of GTP gamma S induced a marked decrease in the number of receptors (from 1.76 fmol/mg protein to 0.78 fmol/mg protein) without significantly altering the dissociation constant (Kd). These results provide evidence that bombesin receptors are coupled to a G-protein signal transduction pathway in guinea pig lung. We have further characterised this G-protein on the basis of its toxin sensitivity. Pretreatment of the lung membranes with either pertussis (10 micrograms/ml) or cholera toxin (50 micrograms/ml) was performed. Cholera toxin treatment did not affect the ability of GTP gamma S to inhibit [125I-Tyr4]bombesin binding to guinea pig lung membranes. However, pertussis toxin treatment induced a decrease in binding and resulted in the inability of GTP gamma S to inhibit [125I-Tyr4]bombesin binding in a concentration-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Toxina da Cólera/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Nucleotídeos de Guanina/farmacologia , Toxina Pertussis , Receptores da Bombesina/metabolismo , Fatores de Virulência de Bordetella/farmacologia , Animais , Autorradiografia , Sítios de Ligação/efeitos dos fármacos , Bombesina/metabolismo , Relação Dose-Resposta a Droga , Proteínas de Ligação ao GTP/efeitos dos fármacos , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacologia , Guanosina Monofosfato/farmacologia , Guanilil Imidodifosfato/farmacologia , Cobaias , Radioisótopos do Iodo , Pulmão/metabolismo , Masculino , Receptores da Bombesina/efeitos dos fármacos , Análise de Regressão , Transdução de Sinais/efeitos dos fármacos , Tionucleotídeos/farmacologiaRESUMO
We have investigated the contractile effect of bradykinin (BK) in guinea pig lung in vitro. BK induces a dose-related contraction of lung parenchymal strips which is increased significantly in the presence of 10(-5) M captopril (an angiotensin converting enzyme inhibitor) or 10(-5) M DL-thiorphan (a neutral endopeptidase inhibitor). The kininase I inhibitor, DL-2-mercaptomethyl-3-guanidino-ethylthiopropionic acid (MGTPA), has no effect on the BK-induced contraction. BK is more potent in contracting parenchymal lung strips than other contractile agents (histamine, carbachol and substance P), however the BK-induced maximal contraction is lower than those obtained with histamine and carbachol. The B1 agonist, des-Arg9-BK, does not contract lung parenchymal strips. The new BK B2 receptor antagonists (Hoe 140, NPC 17731 and NPC 17761), which possess binding affinities in the nanomolar range, inhibit the BK-induced contractile response in a dose-dependent manner. The BK-induced contraction was unaffected by propranolol, atropine, tetrodotoxin, capsaicin pre-treatment, triprolidine, methysergide, Ro 19-3704 and N omega-nitro-L-arginine-methyl-ester (L-NAME), excluding the involvement of nervous pathways, preformed mast cell mediators, platelet-activating factor and nitric oxide. However, indomethacin, a cyclooxygenase inhibitor, AA-861, a 5-lipoxygenase inhibitor, and furegrelate, a thromboxane A2 synthase inhibitor, decreased the contractile response to BK, suggesting that both cyclooxygenase and 5-lipoxygenase products are involved in this contraction.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Bradicinina/farmacologia , Pulmão/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Animais , Bradicinina/antagonistas & inibidores , Bradicinina/metabolismo , Ativação Enzimática , Cobaias , Técnicas In Vitro , Fosfatos de Inositol/metabolismo , Pulmão/fisiologia , Masculino , Inibidores de Proteases/farmacologia , Fosfolipases Tipo C/metabolismoRESUMO
1. Direct ligand binding techniques have been used to compare bradykinin receptors in squamous- or adeno-carcinoma and healthy lung membranes removed from patients during operations. 2. The binding of [3H]-bradykinin to healthy lung membrane is time-dependent and saturable with a KD value of 1.08 +/- 08 nM and a Bmax value of 46.1 +/- 3.1 fmol mg-1 protein (n = 10). In squamous-carcinoma tissue (n = 8) the same amount of receptors are present, Bmax = 52.2 +/- 3.3 fmol mg-1 protein (P = 0.22) but the KD value is significantly higher 2.57 +/- 0.40 nM (P = 0.004). Similar measurements were obtained for adeno-carcinoma tissue (n = 3), KD = 2.80 +/- 0.29 mM (P = 0.001) and Bmax = 49.8 +/- 2.1 fmol mg-1 protein (P = 0.56). 3. In both healthy and squamous-carcinoma preparations, bradykinin analogues displace [3H]-bradykinin binding with the following relative order of potency: Hoe 140 > bradykinin > kallidin > D-Arg0[Hyp3,D-Phe7]bradykinin >>> des-Arg9-bradykinin. Of the analogues used, bradykinin and D-Arg0[Hyp3,D-Phe7]bradykinin appear to be able to differentiate the bradykinin receptors present in both preparations. 4. It is concluded that bradykinin receptors present in healthy and carcinomatous human lung are of the B2 type.
Assuntos
Neoplasias Pulmonares/química , Pulmão/química , Receptores da Bradicinina/análise , Sítios de Ligação , Bradicinina/metabolismo , Humanos , Receptores da Bradicinina/fisiologiaRESUMO
Bradykinin B2 receptor-like binding activity was solubilized from guinea pig lung using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate (Chaps). The binding of [3H]bradykinin to the soluble fraction was time-dependent and saturable. Scatchard analysis of equilibrium binding data indicated that the soluble extract contained a single class of binding sites with a Kd of 696 pM and a Bmax of 57 fmol/mg protein. Unlabelled bradykinin and B2 antagonists inhibited the binding of [3H]bradykinin to Chaps-solubilized extracts with relative potencies similar to those observed with the low-affinity membrane-bound binding sites. Following partial purification of the soluble preparation, using anion exchange (DEAE-Sephacel) and gel filtration (Aca 34) column chromatography steps, two peaks eluted off the column were able to bind [3H]bradykinin and have molecular masses of 168 and 98.5 kDa. The former seems to represent binding of bradykinin to angiotensin converting enzyme (ACE, EC 3.4.15.1) and the latter binding to bradykinin receptor. Using purified commercial ACE, we show that the binding of [3H]bradykinin to ACE can easily be distinguished from that of the bradykinin receptor, since both B1 and B2 ligands were able to inhibit bradykinin binding with affinities clearly different from that expected for a bradykinin receptor.
Assuntos
Pulmão/metabolismo , Peptidil Dipeptidase A/metabolismo , Receptores da Bradicinina/metabolismo , Animais , Sítios de Ligação , Ácidos Cólicos , Cromatografia em Gel , Cromatografia por Troca Iônica , Detergentes , Cobaias , Cinética , Ligantes , Pulmão/enzimologia , Membranas/enzimologia , Membranas/metabolismo , Receptores da Bradicinina/isolamento & purificação , SolubilidadeRESUMO
Bombesin (Bn) and related agonists produce a potent contractile response in guinea pig peripheral airways in vitro, with the following relative potencies: bombesin > gastrin-releasing peptide (GRP) > neuromedin C >> neuromedin B. Specific GRP-preferring receptor antagonists, namely [D-Phe6]Bn-(6-13)methyl ester and [D-Phe6,Cpa14,psi 13-14]Bn(6-14)-NH2, inhibited bombesin-induced lung contraction with high potencies [negative logarithm of the molar concentration of antagonist that produces a twofold shift to the right in the agonist dose-response curve (pA2) of 7.1 and 7.2, respectively], whereas the less-specific antagonist [Leu14,psi 13-14]Bn has a lower one (pA2 of 5.6). In binding studies, the high affinities of GRP, [D-Phe6]Bn(6-13)methyl ester and [D-Phe6,Cpa14,psi 13-14]Bn(6-14)NH2 in contrast with the low affinity of neuromedin B agree with the hypothesis that GRP-preferring receptors are involved in bombesin-induced bronchoconstriction. Bombesin-induced bronchoconstriction is unaffected by atropine, hexamethonium, propranolol, triprolidine, methysergide, Ro 19-3704, and indomethacin or AA-861, suggesting that the Bn response does not occur via a mechanism involving the corresponding endogeneous agents or via the release of arachidonic acid metabolites. Moreover, the effect of Bn is insensitive to capsaicin pretreatment, excluding the involvement of endogeneous neuropeptides. Present results provide evidence that Bn-induced bronchoconstriction results from a direct effect of Bn on bronchial smooth muscle GRP-preferring receptors.
Assuntos
Bombesina/farmacologia , Broncoconstrição , Pulmão/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Receptores de Neurotransmissores/fisiologia , Animais , Bombesina/análogos & derivados , Broncoconstrição/efeitos dos fármacos , Cobaias , Técnicas In Vitro , Ligantes , Masculino , Fragmentos de Peptídeos/farmacologia , Peptídeos/farmacologia , Receptores da Bombesina , Receptores de Neurotransmissores/antagonistas & inibidoresRESUMO
The binding of the radiolabelled bombesin analogue [125I-Tyr4]bombesin to guinea-pig lung membranes was investigated. Binding of [125I-Tyr4]bombesin was specific, saturable, reversible and linearly related to the protein concentration. Scatchard analysis of equilibrium binding data at 25 degrees C indicated the presence of a single class of non-interacting binding sites for bombesin (Bmax = 7.7 fmol/mg protein). The value of the equilibrium dissociation constant (KD = 90 pM) agrees with a high-affinity binding site. Bombesin and structurally related peptides such as [Tyr4]bombesin, neuromedin B and neuromedin C inhibited the binding of [125I-Tyr4]bombesin in an order of potencies as follows: [Tyr4]bombesin greater than bombesin greater than or equal to neuromedin C much greater than neuromedin B. These results indicate that guinea-pig lung membranes possess a single class of bombesin receptors with a high affinity for bombesin and a lower one for neuromedin B.