RESUMEN
PMA enhanced release of the azurophil granule enzyme, beta-glucuronidase, as well as lysozyme, from cytochalasin B-treated PMN's exposed to either zymosan particles or C5a. PMA was active at nanomolar concentrations, was not toxic to the cells, and was most effective when present for brief durations (0-1 min) before exposure of the cells to the stimuli. Beta-glucuronidase was not released in significant amounts from PMN's exposed to PMA alone, in the absence of stimuli such as zymosan or C5a. In contrast, only the specific granule enzyme, lysozyme, was released from unstimulated cells. Electron micrographs of cells exposed to PMA revealed an increase in the number of visible cytoplasmic microtubules as compared to control cells. Enhancement of lysosomal enzyme (beta-glucuronidase) release by PMA appears to be independent of effects on release of specific granule enzymes (lysozyme), but rather is likely due to PMA-induced elevations of cellular cGMP.
Asunto(s)
Glucuronidasa/metabolismo , Lisosomas/metabolismo , Neutrófilos/metabolismo , Forboles/farmacología , Acetato de Tetradecanoilforbol/farmacología , Complemento C5 , Citocalasina B , Humanos , Lisosomas/efectos de los fármacos , Microtúbulos/ultraestructura , Muramidasa/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/ultraestructura , Estimulación Química , ZimosanRESUMEN
Neutrophils stimulated by the chemotactic factor formyl-methionyl-leucyl-phenyl-alanine (FMLP) undergo a transient change in surface properties that permits the cells to adhere more readily to surfaces and to each other. This transient change can be monitored by light scattering as stimulated neutrophils form aggregates while stirred in a platelet aggregometer. Maximum change in light scattering occurs within 1 min and correlates with an increase in the percentage of cells that are in aggregates of four or more cells and a decrease in the percentage of single cells. With time (3-5 min), small aggregates disappear and single cells reappear. The transient change in adhesiveness is accompanied by a persistent change in cell shape; the cells become polarized and protrude ruffles from one sector of the cell surface. During aggregation the cells adhere to one another with smooth sides together and ruffles pointed outward. During disaggregation the cells dissociate laterally with the simultaneous internalization of membrane in the region opposite the ruffles. Particle bound to the surface by charge (thorotrast, cationized ferritin) are concentrated and internalized in this region. The change in cell shape from round to ruffled occurs within seconds, suggesting that membrane is added to the cell surface from an intracellular store. We therefore quantified surface membrane by electron microscopy morphometry and measured a 25% increase within 10 s of adding FMLP. The source of new membrane appeared to be the specific granule membrane since the kinetics of granule discharge (between 30% and 50% of all release occurs in the first 10 s) correlate with the appearance of new membrane. Furthermore, the amount of membrane that appears at the cell surface at 10 s correlates with that lost from intracellular granules in that time. Chemotaxin-induced aggregation thus begins with granule discharge and membrane addition followed by protrusion of ruffles. Adherence is maximal at 60 s and the gradual loss of adhesiveness that follows is associated with uropod formation and enhanced endocytic activity.
Asunto(s)
Quimiotaxis de Leucocito , Neutrófilos/fisiología , Adhesión Celular , Agregación Celular , Membrana Celular/fisiología , Exocitosis , Humanos , Membranas Intracelulares/fisiología , Muramidasa/metabolismo , N-Formilmetionina/análogos & derivados , N-Formilmetionina/farmacología , N-Formilmetionina Leucil-Fenilalanina , Neutrófilos/citología , Neutrófilos/ultraestructura , Oligopéptidos/farmacologíaRESUMEN
Phospholipid remodeling resulting in arachidonic acid (AA) release and metabolism in human neutrophils stimulated by calcium ionophore A23187 has been extensively studied, while data obtained using physiologically relevant stimuli is limited. Opsonized zymosan and immune complexes induced stimulus-specific alterations in lipid metabolism that were different from those induced by A23187. [3H]AA release correlated with activation of phospholipase A2 (PLA2) but not with cellular activation as indicated by superoxide generation. The latter correlated more with calcium-dependent phospholipase C (PLC) activation and elevation of cellular diacylglycerol (DAG) levels. When cells that had been allowed to incorporate [3H]AA were stimulated with A23187, large amounts of labeled AA was released, most of which was metabolized to 5-HETE and leukotriene B4. Stimulation with immune complexes also resulted in the release of [3H]AA but this released radiolabeled AA was not metabolized. In contrast, stimulation with opsonized zymosan induced no detectable release of [3H]AA. Analysis of [3H]AA-labeled lipids in resting cells indicated that the greatest amount of label was incorporated into the phosphatidylinositol (PI) pool, followed closely by phosphatidylcholine and phosphatidylserine, while little [3H]AA was detected in the phosphatidylethanolamine pool. During stimulation with A23187, a significant decrease in labeled PI occurred and labeled free fatty acid in the pellet increased. With immune complexes, only a small decrease was seen in labeled PI while the free fatty acid in the pellets was unchanged. In contrast, opsonized zymosan decreased labeled PI, and increased labeled DAG. Phospholipase activity in homogenates from human neutrophils was also assayed. A23187 and immune complexes, but not zymosan, significantly enhanced PLA2 activity in the cell homogenates. On the other hand, PLC activity was enhanced by zymosan and immune complexes. Stimulated increases in PLC activity correlated with enhanced superoxide generation induced by the stimulus.
Asunto(s)
Ácidos Araquidónicos/sangre , Calcimicina/farmacología , Neutrófilos/metabolismo , Fosfolípidos/sangre , Adulto , Ácido Araquidónico , Humanos , Técnicas In Vitro , Cinética , Neutrófilos/efectos de los fármacos , Fosfolipasas/sangre , Tritio , UltrasonidoRESUMEN
Thyrotropin-releasing hormone (TRH) stimulates prolactin release and (45)Ca(2+) efflux from GH(3) cells, a clonal strain of rat pituitary cells. Elevation of extracellular K(+) also induces prolactin release and increases (45)Ca(2+) efflux from these cells. In this report, we distinguish between TRH and high K(+) as secretagogues and show that TRH-induced release of prolactin and (45)Ca(2+) is independent of the extracellular Ca(2+) concentration, but the effect of high K(+) on prolactin release and (45)Ca(2+) efflux is dependent on the concentration of Ca(2+) in the medium. The increment in (45)Ca(2+) efflux induced by 50 mM K(+) during perifusion was reduced in a concentration-dependent manner by lowering extracellular Ca(2+) from 1,500 to 0.02 muM (by adding EGTA), whereas 1 muM TRH enhanced (45)Ca(2+) efflux similarly over the entire range of extracellular Ca(2+) concentrations. Although 50 mM K(+) caused release of 150 ng prolactin from 40 x 10(6) GH(3) cells exposed to 1,500 muM Ca(2+) (control), reduction of extracellular Ca(2+) to 2.8 muM decreased prolactin release caused by high K(+) to <3% of controls and no prolactin release was detected after exposure to 50 mM K(+) in medium with 0.02 muM free Ca(2+). In contrast, TRH caused release of 64 ng of prolactin from 40 x 10(6) GH(3) cells exposed to medium with 1,500 muM Ca(2+), and release caused by TRH was still 50 and 35% of control in medium with 2.8 and 0.02 muM Ca(2+), respectively. Furthermore, TRH transiently increased by 10-fold the fractional efflux of (45)Ca(2+) from GH(3) cells in static incubations with 1,500 or 3.5 muM Ca(2+), hereby confirming that the enhanced (45)Ca(2+) efflux caused by TRH in both low and high Ca(2+) medium was not an artifact of the perifusion system.Data obtained with chlortetracycline (CTC), a probe of membrane-bound Ca(2+), were concordant with those obtained by measuring (45)Ca(2+) efflux. Cellular fluorescence of CTC varied with the extracellular Ca(2+) concentration and the duration of incubation. TRH decreased the fluorescence of cell-associated CTC in a manner strongly suggesting stimulus-induced mobilization of Ca(2+), and this effect was still demonstrable in GH(3) cells incubated in 50 mM K(+). These data suggest that TRH acts to mobilize sequestered cell-associated Ca(2+) reflected as a (45)Ca(2+) efflux which is independent of the extracellular Ca(2+) concentration. Mobilization of sequestered Ca(2+) into the cytoplasm may elevate free intracellular Ca(2+) and serve to couple stimulation by TRH to secretion of prolactin.
Asunto(s)
Calcio/metabolismo , Hipófisis/metabolismo , Prolactina/metabolismo , Hormona Liberadora de Tirotropina/farmacología , Animales , Calcio/análisis , Calcio/farmacología , Membrana Celular/metabolismo , Células Cultivadas , Clortetraciclina , Potasio/farmacología , Ratas , Espectrometría de FluorescenciaRESUMEN
The effects of iron deficiency and iron overloading on the mitochondrial enzymes involved in heme synthesis were studied in rat livers. The in vitro activities of several of the enzymes in this pathway were differentially influenced by the in vivo iron status of the animals. delta-Aminolevulinic acid synthase was slightly increased in iron-overloaded animals, but remained normal in iron-deficient animals (0.58 +/- 0.09, 0.91 +/- 0.19 and 0.61 +/- 0.12 nmol delta-aminolevulinic acid/mg per h). Copro- and protoporphyrinogen oxidase activities were increased (20 and 60% above controls) in iron-deficient animals. In contrast, coproporphyrinogen oxidase was decreased by 20%, while protoporphyrinogen oxidase remained unchanged in iron-overloaded rats. These variations of activities were not due to changes in the affinity of these enzymes toward their substrates, as coporphyrinogen had the same Km in each case (0.62 +/- 0.05 M) as did protoporphyrinogen (0.22 +/- 0.035 M). Thus, the Km did not vary with the treatment received by the animals. Ferrochelatase activity was measured by both the pyridine hemochromogen method and by measurement of zinc protoporphyrin with endogenous zinc as substrate. In all cases, ferrochelatase was found to be able to synthesize zinc protoporphyrin with endogenous zinc as substrate. However, the apparent Km of zinc chelatase for protoporphyrin was significantly different in the three groups of animals with Km,appProto, app = 2.4 +/- 0.1 10(-7), 4 +/- 0.3 10(-7) and 9.10 +/- 0.05 10(-7) M in iron-overloaded, control and iron-deficient animals, respectively. When ferrochelatase activity was measured by pyridine hemochromogen, identical results were observed in iron-deficient and control animals but decreased by 45% in iron-overloaded animals. The mitochondrial heme content was also decreased by 40% in iron-overloaded rats but unchanged in either iron-deficient or control rats.
Asunto(s)
Hemo/biosíntesis , Hierro/farmacología , Mitocondrias Hepáticas/enzimología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , 5-Aminolevulinato Sintetasa/metabolismo , Animales , Fraccionamiento Celular , Coproporfirinógeno Oxidasa/metabolismo , Ferroquelatasa/metabolismo , Deficiencias de Hierro , Cinética , Masculino , Microscopía Electrónica , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/ultraestructura , Oxidorreductasas/metabolismo , Protoporfirinógeno-Oxidasa , Ratas , Ratas EndogámicasRESUMEN
Primary monolayer cultures of cells derived by enzymatic dispersion from mouse pituitary thyrotropic tumors were demonstrated to be comprised of at least 2 cell types and were separated by selective culture techniques. Suspension cultures of spherical cells had the morphological characteristics of thyrotrophs and produced large quantities of TSH. Separated monolayer cultures of spreading, planar cells produced collagen but almost no TSH. These observations indicate that primary monolayer cultures of cells derived from thyrotropic tumors are heterogeneous, containing only a small proportion of thyrotrophs.
Asunto(s)
Neoplasias Hipofisarias/fisiopatología , Hormona Liberadora de Tirotropina/fisiología , Animales , Células Cultivadas , Colágeno/metabolismo , Femenino , Ratones , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Neoplasias Hipofisarias/metabolismo , Tiroidectomía , Tirotropina/metabolismoRESUMEN
Multilamellar vesicles (MLVs) have been used as drug carriers to increase efficacy or decrease toxicity of a variety of therapeutic agents, including antineoplastics, antibiotics, and immunomodulators. Although analysis of the disposition of encapsulated materials is relatively simple using radiolabels or single enzymes, determining the cellular and subcellular disposition of intact MLVs, i.e., those that still retain their encapsulated materials, is much less straightforward. We have developed a technique that allows demonstration of the uptake of intact MLVs by Kupffer cells. The method is based on co-localization of paired enzymes, glucose oxidase (GO), and horseradish peroxidase (HRP). The rationale for the localization is that H2O2 generated from glucose and oxygen by GO acts as the substrate for the HRP-mediated oxidative polymerization of diaminobenzidine. Therefore, only sites of co-localization of GO and HRP should stain. Mice were injected IV with phosphatidyl choline MLVs encapsulating HRP and GO. Encapsulated enzymes were separated from non-encapsulated by passing the MLVs over a Sepharose 2B column. Control mice were injected with equivalent amounts of free GO. Mice were sacrificed 30 min after injection and liver tissue was fixed in 3% cacodylate-buffered glutaraldehyde for at least 18 hr. Tissues were washed in buffer, then stained in medium containing glucose, diaminobenzidine HCl, and dimethylsulfoxide in 0.1 M cacodylate buffer. In animals injected with MLV-encapsulated GO and HRP, vacuoles in Kupffer cells and some endothelial cells contained electron-dense reaction product. No other cell type, including polymorphonuclear leukocytes, was stained. In control animals no staining was seen. Our results indicate that encapsulation of paired enzymes may be a feasible method to demonstrate the cellular and subcellular disposition of intact liposomes.
Asunto(s)
Histocitoquímica/métodos , Liposomas/análisis , Hígado/análisis , 3,3'-Diaminobencidina/metabolismo , Animales , Dimetilsulfóxido , Portadores de Fármacos , Femenino , Glucosa Oxidasa/administración & dosificación , Glucosa Oxidasa/metabolismo , Peroxidasa de Rábano Silvestre/administración & dosificación , Peroxidasa de Rábano Silvestre/metabolismo , Inyecciones Intravenosas , Liposomas/metabolismo , Hígado/citología , Hígado/metabolismo , Ratones , Ratones Endogámicos BALB C , Microscopía ElectrónicaRESUMEN
The uptake and internalization of tissue-type plasminogen activator (t-PA) by freshly isolated rat hepatocytes was investigated. Electron microscopic examination of the uptake of t-PA-colloidal gold conjugates (t-PA-gold) by isolated rat hepatocytes showed that t-PA-gold was internalized via coated pits. This was inhibited with excess t-PA. Uptake of 125I-t-PA by isolated rat hepatocytes was a rapid, saturable, and specific process. The initial rate of specific uptake was 0.1 fmol/10(6) cells per min. The specific uptake plateaued at 1.4 fmol/10(6) cells by 30 min and declined to 0.8 fmol/10(6) cells at 2 h. Depletion of cellular ATP by 85-90% did not affect the initial rate of specific uptake. However, specific uptake by ATP-depleted hepatocytes at 30 min was reduced by 37%. By 2 h specific uptake by ATP-depleted hepatocytes was only 5% lower than by untreated hepatocytes, suggesting that processing of t-PA and/or its receptor is ATP-dependent. Uptake of 125I-t-PA was temperature dependent. Specific uptake was reduced by approximately 20% at 22 degrees C and by 70% at temperatures below 16 degrees C. Finally, inhibition of coated pit formation by K(+)-depletion with nigericin decreased the uptake of 125I-t-PA. This inhibition was shown to be K(+)-specific since treatment with nigericin in the presence of K+ did not inhibit coated pit formation or 125I-t-PA uptake. A threshold K(+)-depletion level for inhibition of coated pit formation was also demonstrated since treatment under conditions that reduced cellular K+ by only 54% had no effect on coated pit formation or 125I-t-PA uptake. These data support our hypothesis that internalization of t-PA by isolated rat hepatocytes is via coated pits and suggest that uptake of t-PA is a receptor-mediated process.
Asunto(s)
Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Endosomas/metabolismo , Hígado/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Naranja de Acridina , Adenosina Trifosfato/metabolismo , Animales , Inmunohistoquímica , Técnicas In Vitro , Radioisótopos de Yodo , Cinética , Microscopía Electrónica , Potasio/metabolismo , Ratas , Proteínas Recombinantes/metabolismo , TemperaturaRESUMEN
Many stimuli induce neutrophils to undergo an oxidative burst and generate toxic oxygen metabolites. The major products are O2- and H2O2, the latter being presumed to arise by spontaneous dismutation of the former. If H2O2 were indeed derived exclusively from released O2- according to the equation 2O2- + 2H+----H2O2 + O2, one would expect that relationship to be reflected in the ratio of the two metabolites detectable in the extracellular mileu of stimulated neutrophils. A second corollary is that H2O2 should not form when cytochrome c is present to scavenge O2- before it can dismutate. Although H2O2 cannot be measured directly in the presence of cytochrome c because it is consumed in reoxidizing reduced cytochrome c, its presence can be detected indirectly by the ability of catalase to improve the apparent yield of reduced cytochrome c. We found that the relative amounts of extracellular H2O2 and O2- that could be measured in the environment of stimulated neutrophils varied with the stimulus and that catalase protected reduced cytochrome c from H2O2 oxidation when some stimuli were used but not with others. For example, the ratio of O2- to H2O2 produced by neutrophils exposed to PMA was about 2:1, the expected result if H2O2 were derived from O2-. However when cytochalasin B was added to the cells before the stimulus, the yield of H2O2 was reduced but not the yield of O2-. When cells were allowed to settle and spread on tissue culture plastic they produced equimolar amounts of O2- and H2O2. Coating the plastic with IgG doubled cytochrome c reduction without effecting H2O2. In contrast, coating with albumin reduced H2O2 without effecting cytochrome c reduction. Soluble IgG aggregates induced production of mostly O2- whereas immune complexes resulted in release of both metabolites. FMLP and A23187 were similar to the soluble IgG aggregates in their effects and induced release of proportionately more O2- than H2O2. The addition of catalase to the cytochrome c solution improved the yield of reduced cytochrome c when PMA or IgG was used to stimulate the cells but not when FMLP was used. These and other data suggest that H2O2 release is not a linear function of the amount of O2- generated and that either a variable fraction of O2- spontaneously dismutates to H2O2 or the neutrophil NADPH oxidase, in a manner analogous to xanthine oxidase, is capable, under some circumstances, of producing H2O2 as well as O2-.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Peróxido de Hidrógeno/metabolismo , Neutrófilos/metabolismo , Superóxidos/metabolismo , Complejo Antígeno-Anticuerpo , Catalasa/farmacología , Ensayo de Inmunoadsorción Enzimática , Histocitoquímica , Humanos , Inmunoglobulina G/farmacología , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/citología , Fagocitosis/efectos de los fármacos , Estimulación Química , Superóxido Dismutasa/farmacología , Acetato de Tetradecanoilforbol/farmacología , Factores de TiempoRESUMEN
Radiolabeled human peripheral blood monocytes released [3H]arachidonic acid upon challenge with the calcium ionophore A23187 (10 microM), or f-Met-Leu-Phe (FMLP, 1 microM). Chromatographic analysis of [3H]arachidonic acid labeled phospholipids showed that stimulation by FMLP reduced the amount of labeled phosphatidylcholine exclusively. Treatment of the monocytes with 10(-3) M dibutyryl cyclic AMP (d-cAMP) or 5 X 10(-4) M isobutylmethylxanthine (IBMX) substantially inhibited [3H]arachidonic acid release (30%) and depletion from labeled phosphatidylcholine (PC) in FMLP--but not calcium ionophore--stimulated cells. Using the fluorescent probe Indo-1, the FMLP-induced cytosolic calcium increase was unaffected by 10(-3) M dibutyryl cyclic AMP. The results suggest that FMLP-stimulated phospholipase activity is regulated by cyclic AMP, but not by depressing receptor-medicated increases in cytoplasmic free calcium.
Asunto(s)
Ácidos Araquidónicos/metabolismo , Calcio/metabolismo , AMP Cíclico/fisiología , Monocitos/efectos de los fármacos , N-Formilmetionina Leucil-Fenilalanina/farmacología , Fosfolípidos/metabolismo , 1-Metil-3-Isobutilxantina/farmacología , Ácido Araquidónico , Bucladesina/farmacología , Calcimicina/farmacología , Células Cultivadas , Cromatografía en Capa Delgada , Citosol/efectos de los fármacos , Citosol/metabolismo , Humanos , Monocitos/metabolismoAsunto(s)
Neutrófilos/metabolismo , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/análogos & derivados , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , Aniones , Complejo Antígeno-Anticuerpo/metabolismo , Sitios de Unión/efectos de los fármacos , Transporte Biológico/efectos de los fármacos , Humanos , Canales Iónicos/efectos de los fármacos , Canales Iónicos/metabolismo , Neutrófilos/efectos de los fármacos , Sulfatos/metabolismoRESUMEN
The random migration of neutrophils under agarose as measured by the number of cells leaving the well, is enhanced when the pH or the osmolality of the medium is reduced or when microtubule agents are used. Concentrations of colchicine above 5 x 10-7 M increased the number of cells migrating and decreased the mean number of centriolar microtubules in a dose-dependent fashion from 16 to 4 per 4 micron 2 at 10-5 M. The distance that colchicine-treated neutrophils migrated from the well was not different from the control. Lowering the pH from 7.4 to 6.0 also increased random migration and decreased pericentriolar microtubules from a mean of 16 to a mean of 10. At pH 6.0, both the number of cells that migrated and the distance the cells forming the leading edge travelled from the well were increased. Since peripheral microtubules may play a greater role in cell migration than centiolar ones, we examined the numerical density of microtubules in the peripheral cytoplasm. Lowering the medium pH reduced the mean number of microtubules per 10 micron 2 from 6 to 2. Colchicine reduced micro-tubules in the same area to I. At the low pH, colchicine reduced even further the numbers of both centriole-associated and peripheral microtubules but the migration pattern was the same as that seen at pH 6.0 without colchicine. Lowering medium osmolality from 280 to 230 m-osmol increased random migration but did not affect microtubule numbers. The addition of colchicine to this system decreased microtubule numbers and increased migration even further. Conditions that enhanced neutrophil migration also affected cell shape. Whereas cells at pH 7.4 were generally fan-shaped with a broad, smooth leading edge, cells at pH 6.0 with or without colchicine were long and narrow. Neutrophils at pH 7.4 but 230 m-osmol had a scalloped edge, which often appeared thickened. This too was not altered by colchicine. The morphology of cells treated with colchicine was similar to controls except for the more frequent presence of long retraction fibres. Each of these treatments thus appears to act on a different aspect of the cell's locomotory apparatus. The mechanisms by which colchicine and lowered ph enhance migration may partially overlap since both significantly decrease peripheral microtubules. The data suggest that microtubules play a constraining role within the cell, limiting the ability of the cell to move and change direction.
Asunto(s)
Microtúbulos , Neutrófilos/fisiología , Adulto , Movimiento Celular/efectos de los fármacos , Colchicina/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Concentración de Iones de Hidrógeno , Microscopía Electrónica , Microtúbulos/efectos de los fármacos , Microtúbulos/ultraestructura , Neutrófilos/ultraestructura , Concentración OsmolarRESUMEN
The complement component, C5a provokes the selective release of granule-associated enzymes from the intact, viable cytochalasin B-treated human polymorphonuclear leukocytes (PMN) in the absence of phagocytosis or cellular adherence to surfaces. Consquently, in this experimental system the influence of divalent cations on these two processes can be disregarded and their effects on enzymes secretion can be studied directly. Cytochalasin B-treated PMN exposed to C5a in calcium and magnesium-free media consistently secreted significant amounts of the granule-associated enzymes, beta-glucuronidase and lysozyme. The basal secretory response was not diminished if cells were preincubated with 5.0 mM EDTA, nor was it influenced if 1.0 mm or 2.0 mM EDTA were present in the reaction mixtures. The addition of calcium (up to 1.5 to 2.0 mM) produced a concentration-dependent enhancement of beta-glucuronidase release, whereas increasing amounts of calcium (above 2.0 mM) inhibited secretion of this enzyme. Lysozyme release was similarly enhanced by the addition of calcium, but inhibition with high concentrations was not observed. Calcium per se, in the absence of C5a, provoked only the release of lysozyme from these cells. The effects of calcium upon enzyme release were not associated with alterations in the state of assembly of cytoplasmic microtubules. These findings provide another example of the role of calcium in "stimulus-secretion coupling" and provide evidence that exocytosis of various granules in human PMN is regulated by independent mechanisms involving calcium.
Asunto(s)
Cationes Bivalentes/farmacología , Complemento C5 , Proteínas del Sistema Complemento , Glucuronidasa/metabolismo , Granulocitos/enzimología , Leucocitos/enzimología , Muramidasa/metabolismo , Adulto , Calcio/farmacología , Adhesión Celular , Citocalasina B , Dimetilsulfóxido , Glucuronatos/metabolismo , Granulocitos/inmunología , Humanos , L-Lactato Deshidrogenasa/análisis , Leucocitos/ultraestructura , Micrococcus/metabolismo , Microscopía Electrónica , Microtúbulos , Fagocitosis , FenolftaleínasRESUMEN
Rats were chronically iron-overloaded by intraperitonel injections of iron-dextran. Electron microscopy revealed that the excess iron was deposited in ferritin-like particles packed in lysosomes and scattered in hepatic cytoplasm. No mitochondrial iron deposition or damage was seen. Furthermore, mitochondrial preparations from chronically iron-overloaded animals were found to be contaminated with lysosomes, which could explain previously reported increases in mitochondrial iron by chemical analysis. Mitochondrial function, as measured by cytochromes a-a3, b and c concentrations as well as activity of the rate-limiting enzyme of haem synthesis, delta-aminolaevulinate synthetase, was not diminished by chronic iron-overloading. Microsomal haem was decreased by 30% at the time that haem oxygenase, the rate-limiting enzyme of haem degradation, was increased approx. 3-fold. Animals were given a single intraperitoneal injection of iron-dextran and the activities of delta-aminolaevulinate synthetase and haem oxygenase were measured over 24 h. delta-Aminolaevulinate synthetase activity increased approx. 2-fold in these acutely iron-overloaded rat livers, but at a time after the increase in haem oxygenase. These results suggest that an early consequence of excess iron in liver is acceleration of the rate of haem degradation, possible by haem oxygenase.
Asunto(s)
Hierro/farmacología , Hígado/enzimología , Oxigenasas/biosíntesis , 5-Aminolevulinato Sintetasa/metabolismo , Animales , Citocromos/metabolismo , Inducción Enzimática/efectos de los fármacos , Hemo/metabolismo , Hígado/efectos de los fármacos , Hígado/ultraestructura , Masculino , Microsomas Hepáticos/metabolismo , RatasRESUMEN
Tritiated arachidonic acid (3H-AA)-labeled rat synovial fibroblasts stimulated with human recombinant interleukin-1 beta (rIL-1 beta) released incorporated radiolabel in a time-dependent and dose-dependent manner, with labeled prostaglandins representing 29% of the released radiolabel. Treatment of the cells with dibutyryl cAMP or prostaglandin E2 enhanced both spontaneous and rIL-1 beta-induced 3H-AA release; treatment with indomethacin or naproxen inhibited the response. The effects of these cyclooxygenase inhibitors on 3H-AA release were not reversed by the addition of prostaglandin E2. The activities of phospholipase A, phospholipase C, and diglyceride lipase were detected in the homogenates of rat synovial fibroblasts. Pretreatment of synovial cells with rIL-1 beta resulted in a threefold stimulation of phospholipase A activity and a slight increase in phospholipase C activity in cell homogenates. These data show that rIL-1 beta stimulates phospholipase activities in rat synovial fibroblasts and that at least one of these activities may be regulated by either prostaglandins or cAMP.
Asunto(s)
Interleucina-1/farmacología , Fosfolipasas/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Membrana Sinovial/enzimología , Animales , Ácido Araquidónico , Ácidos Araquidónicos/metabolismo , Bucladesina/farmacología , AMP Cíclico/metabolismo , Dinoprostona/farmacología , Activación Enzimática , Fibroblastos/enzimología , Ratas , Ratas Endogámicas Lew , Proteínas Recombinantes/farmacología , Membrana Sinovial/patología , Distribución TisularRESUMEN
Generation of toxic oxygen metabolites by inflammatory cells is considered to be one of the mechanisms by which inflammation produces tissue injury. This concept is based on in vitro studies of purified leukocyte populations because it has not been possible to assess production of these metabolites in tissues. In order to determine whether or not inflammatory cells in tissue generate H2O2, the authors modified an earlier cytochemical method for the localization of H2O2. The incubation medium consists of 0.5 mM CeCl3 in a Hepes-buffered balanced salt solution with Cl- as the only anion. Synovial tissue from the knees of normal and 16-day adjuvant arthritic rats was incubated in this medium for 30 minutes and then fixed and processed for electron microscopy. No H2O2 reaction product was visible in normal synovium. In contrast, patchy deposits of H2O2 reaction product were seen adjacent to a subpopulation of synovial lining macrophages in synovium from inflamed knee joints. These data show that rat synovial macrophages are capable of generating H2O2 when appropriately stimulated and that such a stimulus is present in adjuvant arthritis.
Asunto(s)
Artritis Experimental/metabolismo , Artritis/metabolismo , Peróxido de Hidrógeno/metabolismo , Macrófagos/metabolismo , Membrana Sinovial/metabolismo , Animales , Artritis Experimental/patología , Macrófagos/ultraestructura , Masculino , Microscopía Electrónica , Ratas , Ratas Endogámicas Lew , Membrana Sinovial/ultraestructuraRESUMEN
Stimulation of [3H]-arachidonic acid labeled human synovial cells with 3.0 X 10(-10)M recombinant interleukin-1 or tumor necrosis factor resulted in the release of incorporated radiolabel (41.1% and 27.7% respectively). Analysis of [3H]-arachidonic acid labeled phospholipids showed that interleukin-1 and tumor necrosis factor diminished [3H]-arachidonic acid in phosphatidyl-choline phosphatidylserine and phosphatidylinositol. Treatment of [3H]-arachidonic acid labeled cells with 10(-3)M dibutyryl cyclic AMP or 10(-6)M PGE2 did not affect spontaneous or stimulated [3H] arachidonic acid release significantly. These data show that recombinant interleukin-1 and tumor necrosis factor stimulate human synovial cell phospholipase activity in a similar manner and that this activity is not affected significantly by agents that elevate cyclic AMP.
Asunto(s)
Ácidos Araquidónicos/metabolismo , Glicoproteínas/farmacología , Interleucina-1/farmacología , Fosfolípidos/metabolismo , Membrana Sinovial/metabolismo , Ácido Araquidónico , Bucladesina/farmacología , Células Cultivadas , Dinoprostona , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Humanos , Fosfolipasas A/metabolismo , Prostaglandinas E/farmacología , Proteínas Recombinantes/farmacología , Factores de Tiempo , Factor de Necrosis Tumoral alfa , Fosfolipasas de Tipo C/metabolismoRESUMEN
Human neutrophils in suspension undergo a metabolic burst and generate reactive O2- metabolites upon exposure to many soluble and particulate stimuli. They can also be stimulated to produce O2- when in contact with surfaces. We found that when neutrophils were allowed to settle into protein-coated surfaces the amount of O2- they generated varied with the nature of the protein: IgG greater than bovine serum albumin greater than plastic greater than gelatin greater than serum greater than collagen. However, when polymorphonuclear leukocytes were permitted to settle onto a surface and then were stimulated with either phorbol myristate acetate or N-formyl-methionyl-leucyl-phenylalanine the O2- response was greatly diminished compared to control cells that were exposed to the stimulus in suspension. In contrast, superoxide production in response to the particulate stimulus opsonized zymosan was similar in both suspended and settled neutrophils. The degree of inhibition was not related to the degree of adherence since the diminished response occurred with all of the surfaces tested and in the presence of cytochalasin B. Onset of inhibition was very rapid as was recovery when cells were resuspended. Whereas production of O2- was greatly inhibited by surface contact, release of lysosomal enzymes was only slightly affected. The effect of surface contact did not appear to be mediated via activation of adenylate cyclase since the combination of a phosphodiesterase inhibitor and exogenous dibuteryl cyclic adenosine monophosphate did not inhibit phorbal myristate acetate O2- production, but surface contact did. These data indicate that surface contact such as would occur during diapedesis and chemotaxis profoundly alters neutrophil behavior by an unknown mechanism and imply that observations made on polymorphonuclear leukocytes in suspension cannot be generalized to polymorphonuclear leukocytes in tissue.
Asunto(s)
Neutrófilos/metabolismo , Superóxidos/metabolismo , Adhesión Celular , AMP Cíclico/metabolismo , Citocalasina B/farmacología , Humanos , Peróxido de Hidrógeno/metabolismo , Técnicas In Vitro , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/efectos de los fármacos , Neutrófilos/ultraestructura , Propiedades de Superficie , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
We studied the capacities of naked and protein-coated monosodium urate (MSU) crystals to stimulate superoxide anion (O(2)) release by human polymorphonuclear leukocytes (PMN). Uncoated MSU estimated significant O(2) production by cytochalasin B-treated PMN. Precoating MSU with IgG caused an increase in mean O(2) production, whereas precoating heated MSU with serum or plasma inhibited O(2) release. Unheated MSU crystals, which activate complement to a greater extent than heated crystals, also provoked O(2) generation, an effect again abrogated by precoating with serum but not with plasma. Coincubation of unheated MSU and plasma resulted in an enhancement of O(2) generation. The results of these experiments support the hypothesis that adsorbed proteins modulate the phlogistic potential of MSU and that the surface activation of humoral mediators contributes to the local inflammatory response.
Asunto(s)
Activación de Complemento/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Oxígeno/biosíntesis , Proteínas/metabolismo , Superóxidos/biosíntesis , Ácido Úrico/farmacología , Adsorción , Complemento C5/metabolismo , Cristalización , Humanos , Inmunoglobulina G/metabolismo , Técnicas In Vitro , Inflamación/metabolismo , Superóxidos/metabolismoRESUMEN
Although adherence to surfaces is central to neutrophil function many of the determinants of neutrophil adherence are still unknown. The possible involvement of cell surface material, fibronectin in particular, was therefore studied. Surface coat material was visualized ultrastructurally by the ferrocyanide--reduced osmium technique of Karnovsky (1971). Loosely attached surface coat material was seen distributed uniformly on cells in suspension. Indirect immunofluorescence indicated the presence of fibronectin on the neutrophil surface. Distribution of fibronectin as determined by indirect immunoferritin localization corresponded with the distribution of cell coat material. Some, if not all, of this fibronectin was synthesized by neutrophils themselves since metabolically labelled fibronectin could be obtained by immunoprecipitation after short-term culture with [36S]methionine. Neutrophils also adhere to Sepharose beads to which gelatin is covalently linked (GS) but not to plain Sepharose beads (PS). In the process they transfer surface coat material to GS but not PS. Similar transfer was seen when cells were permitted to adhere to glass or plastic coverslips. Indirect immunofluorescence showed that fibronectin-containing material was transferred from neutrophils to GS but not PS. Parallel studies with antisera to 2 other plasma proteins, factor VIIIR and alpha 1-antitrypsin showed that neutrophils did not transfer these to either GS or PS beads. The data suggest that material antigenically and functionally related to fibronectin is associated with the extracellular coat of neutrophils and is transferred with cell surface material to surfaces to which neutrophils adhere.