RESUMO
Binding of the dihydropyridine calcium antagonist PN200-110 was studied in human myometrial membranes. PN200-110 bound reversibly and with high affinity to membrane fragments. The highest concentration of binding sites was found in the sarcolemma. The benzothiazepine calcium antagonist diltiazem stimulated PN200-110 binding by increasing the amount bound at equilibrium. Kinetic studies detected a fast and slow rate of dissociation in the presence of diltiazem.
Assuntos
Bloqueadores dos Canais de Cálcio/metabolismo , Diltiazem/farmacologia , Miométrio/metabolismo , Oxidiazóis/metabolismo , Sarcolema/metabolismo , Fracionamento Celular , Feminino , Humanos , Isradipino , Cinética , Frações Subcelulares/metabolismoRESUMO
Membrane fragments were prepared from nonpregnant human myometrium and fractionated by differential and sucrose gradient centrifugation. Reversible high-affinity binding sites of the dihydropyridine calcium antagonist nitrendipine were identified in highest density in that membrane fraction most enriched in markers for plasmalemma. In determinations done on several preparations, the equilibrium dissociation constant for nitrendipine at 37 degrees C was found to be 0.3 to 0.8 nM and was the same for each fraction. The maximum binding capacity was found to be 400-500 fmol/mg protein in the plasmalemma fraction.
Assuntos
Canais Iônicos/metabolismo , Miométrio/metabolismo , Nifedipino/análogos & derivados , Cálcio/metabolismo , Fracionamento Celular , Membrana Celular/metabolismo , Feminino , Humanos , Nifedipino/metabolismo , NitrendipinoRESUMO
Like in the polymorphonuclear leukocyte (PMN), the platelet-derived growth factor (PDGF) purified to homogeneity is capable of inducing monocyte activation responses as evaluated by generation of superoxide anion (O-.2) from membrane-associated oxidase system, release of granule enzymes, and enhanced cell adherence and cell aggregation. Superoxide anion release was maximized at 10 ng/mL PDGF and was comparable to that induced by 10(-7) mol/L formyl-methionyl-leucyl-phenylalanine. The potency of PDGF to induce this response in monocytes was of the same magnitude as that observed in PMNs. Similarly, lysozyme release and monocyte adherence were also increased in a dose-dependent manner and achieved maximal responses at 40 ng/mL concentration of PDGF. The PDGF concentration required to achieve maximal monocyte aggregation was two-fold (60 ng/mL) of that found for PMNs. In contrast to PMNs, a positive correlation (gamma = .93; P less than .01) was observed between the increases of PDGF concentration and beta-glucuronidase release. These findings indicate that PDGF can induce the full sequence of cell activation events in human monocytes similar to human PMNs.
Assuntos
Monócitos/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/farmacologia , Adesão Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Técnicas In Vitro , Lisossomos/enzimologia , Neutrófilos/efeitos dos fármacos , Superóxidos/metabolismoRESUMO
The platelet-derived growth factor (PDGF) has several well defined important biologic activities. Platelet-derived growth factor is the major mitogen in human serum for cells of mesenchymal origins; it is a potent chemoattractant protein for human monocytes, neutrophils, fibroblasts, and smooth muscle cells; and has been implicated in transformation by simian sarcoma virus and perhaps in transformation by other agents as well. In this article, PDGF has been shown to stimulate activation of human peripheral blood neutrophils defined by loss of membrane associated calcium as reflected by loss of chlortetracycline fluorescence, release of superoxide anion and specific granule enzymes, and enhanced neutrophil adherence and aggregation. These responses occurred in a dose-dependent fashion at concentrations of PDGF between 10 ng/mL (0.4 nmol/L) and 40 ng/mL (1.5 nmol/L) and were comparable to effects obtained with optimal concentrations of fMLP and C5a. Degranulation induced by PDGF was selective for secondary (specific) granules and not primary (azurophil) granules. Platelet-derived growth factor thus is ideally suited for a pivotal role in attracting inflammatory cells locally and initiating neutrophil activation at sites of blood vessel injury. Platelet-derived growth factor or a closely related protein also may play an important role in attracting and activating neutrophils in association with inflammatory tumors.
Assuntos
Neutrófilos/fisiologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Cálcio/sangue , Adesão Celular/efeitos dos fármacos , Agregação Celular/efeitos dos fármacos , Clortetraciclina/farmacologia , Grânulos Citoplasmáticos/enzimologia , Relação Dose-Resposta a Droga , Fluorescência , Humanos , Neutrófilos/metabolismo , Fator de Crescimento Derivado de Plaquetas/biossíntese , Superóxidos/metabolismoRESUMO
Human polymorphonuclear neutrophils (PMN) were treated with the antiinflammatory agents dexamethasone or Auranofin. PMN treated with dexamethasone in a dose range of 0.25-1 microM or Auranofin, 5-15 mM, were stimulated with 10(-7)M N-formyl-methionyl-leucyl-phenylalanine (FMLP). These agents were shown to inhibit the functional responses of degranulation and superoxide production in a dose-dependent manner. Similarly, the change in electrophoretic mobility, reflecting cell surface charge, was blocked. While both agents inhibited change in the fluorescence of the calcium chelate probe chlorotetracycline (CTC), the pattern of inhibition was significantly different. Dexamethasone appeared to inhibit the CTC response during its latter phases, while Auranofin inhibited all aspects of the CTC response. Auranofin was additionally shown to significantly decrease specific binding of FMLP, as well as the number of FMLP receptors. The two agents thus appear to act by different mechanisms. Dexamethasone is shown to have an effect on membrane-bound calcium release as measured by CTC, while Auranofin interferes with receptor binding.
Assuntos
Aurotioglucose/análogos & derivados , Dexametasona/farmacologia , Neutrófilos/efeitos dos fármacos , Auranofina , Aurotioglucose/farmacologia , Clortetraciclina/farmacologia , Citocalasina B/farmacologia , Grânulos Citoplasmáticos/efeitos dos fármacos , Eletroforese , Fluorescência , Ouro , Humanos , Lactoferrina/metabolismo , Muramidase/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/enzimologia , Receptores de Superfície Celular/análise , Receptores de Formil Peptídeo , Superóxidos/metabolismoRESUMO
Standard isolation techniques for the human polymorphonuclear leukocyte (PMN) involve sequential exposure of cells to the nonphysiologic environments of dextran, Ficoll-Hypaque (FH) gradient centrifugation, and hypotonic conditions. It has been suggested that these may be harmful to the recovered PMN. Counterflow centrifugal elutriation (CCE) allows separation of human PMNs while the cells are continuously bathed in a physiologic and isotonic buffer. To investigate whether preparative technique may alter PMN activation, we compared PMNs obtained by these two methods for stimulus-induced superoxide production and release of primary and specific granule contents. Resting PMN volume was also evaluated. We observed that PMNs obtained using the CCE method were larger and released significantly more superoxide and specific granule contents than PMNs obtained by the standard FH technique. The possible origins for these differences are discussed.
Assuntos
Separação Celular/instrumentação , Grânulos Citoplasmáticos/enzimologia , Neutrófilos/metabolismo , Superóxidos/sangue , Adulto , Sedimentação Sanguínea , Contagem de Células , Centrifugação/métodos , Centrifugação com Gradiente de Concentração , Glucuronidase/sangue , Humanos , Muramidase/sangue , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/citologiaRESUMO
Previous studies have identified patients with susceptibility to bacterial infection associated with lactoferrin deficiency in dysmorphic neutrophils containing abnormal or no secondary granules and abnormal nuclear segmentation. We have investigated the subcellular distribution of vicinal glycol-containing complex carbohydrates in marrow and blood myeloid cells of such a patient using the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining method and have examined the response of these neutrophils to the degranulating agents N-formylmethionyl-leucyl-phenylalanine (FMLP) and phorbol myristate acetate (PMA). As in normal specimens, immature primary granules were strongly PA-TCH-SP reactive; however, unlike normal specimens, masking of PA-TCH-SP reactivity did not occur in mature primary granules. Endoplasmic reticulum demonstrated moderately strong PA-TCH-SP staining, in contrast to absent staining of this organelle in normal promyelocytes and consistent with abnormal primary granule genesis. Small abnormal elongated granules (0.1-0.2 micron in diameter) were identified at the myelocyte state of development and were the predominant granule type in late neutrophils. These granules were identified as secondary granules on the basis of their PA-TCH-SP positivity and were differentiated from primary and tertiary granules on the basis of a lack of peroxidase, acid phosphatase, and sulfate staining. When the neutrophils were exposed to PMA, cell aggregation occurred, and the abnormal granules degranulated in a manner similar to the degranulation observed with normal secondary granules. Although PA-TCH-SP staining of the plasma membrane appeared normal, a decrease in FMLP receptors was demonstrated. Thus, a defect(s) is present in complex carbohydrate distribution and staining that involves primary and secondary granules and possibly the plasmalemma of neutrophils from this patient. This results in abnormal packaging of primary granules and synthesis of normal numbers of secondary granules that are qualitatively and morphologically abnormal, but can be recruited to degranulate with PMA.
Assuntos
Carboidratos/sangue , Lactoferrina/deficiência , Lactoglobulinas/deficiência , Neutrófilos/análise , Fosfatase Ácida , Fosfatase Alcalina/deficiência , Grânulos Citoplasmáticos/análise , Humanos , N-Formilmetionina/análogos & derivados , N-Formilmetionina Leucil-Fenilalanina , Neutrófilos/ultraestrutura , Oligopeptídeos , Peroxidases , Coloração e Rotulagem , Sulfatos , Acetato de TetradecanoilforbolRESUMO
Cetiedil citrate monohydrate inhibits sickling of red cells and aggregation of platelets. We assessed its ability to attenuate polymorphonuclear leukocyte (PMN) function. PMN aggregation in response to 2 X 10(-7) M formyl-met-leu-phe (FMLP) was inhibited in a dose-dependent fashion by cetiedil concentrations ranging from 60 to 250 microM. Additionally, 125 microM cetiedil inhibited PMN aggregation in response to 2 X 10(-7) M FMLP, 20 ng/ml phorbol myristate acetate (PMA), and 1 X 10(-6) M A23187 by 69% +/- 18%, 72% +/- 20%, and 65% +/- 4%, respectively. Inhibition of FMLP-induced aggregation was provided by only 5 min of incubation of the drug with the cells and was partially reversible. Cell viability was unaffected by exposure of PMN to the drug. Correspondingly, 125 microM cetiedil prevented the translocation of calcium from the PMN membrane as assessed by chlorotetracycline fluorescence. Paralleling the effect of the drug on PMN aggregation, 125 microM cetiedil inhibited release of superoxide by 55% and decreased the number of available 3H-FMLP receptors. However, its effect on release of the primary granule constituent, myeloperoxidase, was minimal (4.5% inhibition), while the effect on release of the specific granule product, lactoferrin (27% inhibition), was modest. These studies indicate that cetiedil affects PMN aggregation and superoxide release to a much greater extent than PMN degranulation. Thus, cetiedil may have potential uses in modulating inflammatory response in vivo.
Assuntos
Azepinas/farmacologia , Neutrófilos/efeitos dos fármacos , Sítios de Ligação/efeitos dos fármacos , Cálcio/metabolismo , Agregação Celular/efeitos dos fármacos , Citocalasinas/farmacologia , Humanos , Lactoferrina/sangue , N-Formilmetionina/análogos & derivados , N-Formilmetionina/metabolismo , N-Formilmetionina/farmacologia , N-Formilmetionina Leucil-Fenilalanina , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , Peroxidase/sangue , Superóxidos/metabolismoRESUMO
Two human peripheral blood monocyte subsets and lymphocytes were isolated by counterflow centrifugal elutriation (CCE). The cell volumes of 303 mu3 and 380 mu3 were measured for the smaller and larger monocyte populations, respectively. Superoxide release by large monocytes exposed to opsonized zymosan was five times more active than that of the small monocytes. The production of colony stimulating activity was two-fold greater and the myeloperoxidase activity was 1.4-fold greater by the larger monocytes. Enriched fractions of cytotoxic cells responsible for natural killer (NK) activity against neuroblastoma cells were also obtained by counterflow centrifugal elutriation. Natural killer cells were obtained in larger lymphocyte fractions and had a mean cell volume of 180 mu3. Compared with the NK activity against the neuroblastoma cells, both the small and large monocytes displayed greater antibody-dependent cellular cytotoxicity (ADCC) activity against human erythrocytes. The larger peripheral blood monocytes aggregated in response to FMLP (N-formyl-methionyl-leucyl-phenyl alanine peptide) and PAF (platelet activating factor). Unlike the granulocyte, monocyte aggregation in response to FMLP was not accompanied by degranulation nor was it potentiated by cytochalasin B. In addition, monocyte aggregation could be blocked by benoxaprofen, unlike the granulocyte. Thus, CCE provides a means of isolating subsets of monocytes and lymphocytes and obtaining large numbers of peripheral blood monocytes for functional studies.
Assuntos
Granulócitos/fisiologia , Linfócitos/fisiologia , Citotoxicidade Celular Dependente de Anticorpos , Agregação Celular , Linhagem Celular , Separação Celular , Centrifugação , Ensaio de Unidades Formadoras de Colônias , Eritrócitos/imunologia , Granulócitos/classificação , Humanos , Células Matadoras Naturais/fisiologia , Linfócitos/classificação , Microscopia Eletrônica de Varredura , Neuroblastoma/patologia , Superóxidos/sangueAssuntos
Carbono-Nitrogênio Ligases , Ligases/metabolismo , Neoplasias Hepáticas Experimentais/enzimologia , Animais , Citidina Trifosfato/imunologia , Citidina Trifosfato/metabolismo , Humanos , Imunodifusão , Ligases/imunologia , Neoplasias Hepáticas Experimentais/imunologia , Uridina Trifosfato/imunologia , Uridina Trifosfato/metabolismoAssuntos
Adenocarcinoma/patologia , Neoplasias do Colo/patologia , Animais , Divisão Celular , Neoplasias do Colo/enzimologia , Glicólise , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Pentosefosfatos/metabolismo , Purinas/metabolismo , Pirimidinas/metabolismo , Transplante HeterólogoAssuntos
Antimetabólitos Antineoplásicos/farmacologia , Ciclo Celular/efeitos dos fármacos , Neoplasias Hepáticas Experimentais/patologia , Ribonucleosídeos/farmacologia , Amidas , Animais , Antimetabólitos Antineoplásicos/antagonistas & inibidores , Células Cultivadas , Relação Dose-Resposta a Droga , Hipoxantinas/farmacologia , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Ácidos Nucleicos/metabolismo , Orotidina-5'-Fosfato Descarboxilase/metabolismo , Pirazóis , Nucleosídeos de Pirimidina/metabolismo , Ribose , Uracila/metabolismoAssuntos
Osso e Ossos/metabolismo , Cálcio da Dieta/farmacologia , Fósforo/farmacologia , Esforço Físico , Animais , Osso e Ossos/anatomia & histologia , Cálcio/metabolismo , Relação Dose-Resposta a Droga , Feminino , Fêmur/metabolismo , Camundongos , Fósforo/metabolismo , Tíbia/metabolismo , Fatores de TempoAssuntos
Neoplasias Hepáticas Experimentais/metabolismo , Animais , Divisão Celular , Linhagem Celular , Transformação Celular Neoplásica/metabolismo , Desoxirribonucleosídeos/metabolismo , Guanina Desaminase/metabolismo , Humanos , Neoplasias Hepáticas Experimentais/enzimologia , Camundongos , Modelos Biológicos , Purina-Núcleosídeo Fosforilase/metabolismo , Pirimidinas/metabolismo , Ratos , Inanição/enzimologiaAssuntos
Regulação da Expressão Gênica , Neoplasias Hepáticas Experimentais/genética , Animais , Linhagem Celular , Desoxirribonucleosídeos/metabolismo , Fígado/enzimologia , Neoplasias Hepáticas Experimentais/enzimologia , Neoplasias Hepáticas Experimentais/metabolismo , Nucleosídeos/metabolismo , Ratos , Ribonucleosídeos/metabolismoRESUMO
An experiment was conducted to assess the physiological effects of a diet rich in foods cotaining phosphate additives. During a 4-week control period, eight adults were fed a balanced diet free of phosphate additives providing approximately 95 g protein 0.7 g Ca and 1.0 g P per day. During a subsequent 4-week period, food items containing phosphate additives were substituted for counterpart items devoid of added phosphates. This diet contained 0.7 g Ca and 2.1 g P per day. The introduction of foods containing phosphate additives was associated with intestinal distress, soft stools or mild diarrhea. These symptoms subsided in six subjects but occurred intermittently throughout the experimental period in the other two subjects. The high-phosphorus diet induced increases in serum phosphorus and urinary phosphorus and decreases in serum calcium and urinary calcium. Hydroxyproline excretion in the urine was increased and cyclic AMP excretion was elevated in six of the eight subjects. These changes are analogous to those seen in experimental animals fed high-phosphorus diets which were shown to be due to enhanced parathyroid activity (secondary hyperparathyroidism). The use of phosphate food additives is discussed with respect to their possible stimulating effect on adult bone resorption.