Inhibition Of Enzyme Release Research Articles

Cyclic GMP mediates SIN-1-induced inhibition of human polymorphonuclear leukocytes

Different nitrovasodilators were used to assess the role of cyclic GMP in the regulation of polymorphonuclear leukocyte (PMN) function. Molsidomine and its metabolites, 3-morpholinosydnonimine (SIN-1) and N-nitro-N-morpholinoaminoacetonitrile (SIN-1A) at 0.01–1 mM, inhibited lysosomal enzyme release from PMN stimulated by 30 nM formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP). At 1 mM, molsidomine, SIN-1 and SIN-1A decreased ß-glucuronidase release by 19, 37 and 46% of the control, respectively. Glyceryl trinitrate (GTN) and sodium nitroprusside (SNP) showed no effect on ß-glucuronidase release from PMN. At 1 mM, SIN-1A, SIN-1 and SNP in the presence of 0.5 mM isobutylmethylxanthine (IBMX) stimulated cyclic GMP 21-, 9- and 14-fold, respectively, demonstrating a relation between cyclic GMP stimulation and neutrophil inhibition by the molsidomine metabolites. GTN and unmetabolized molsidomine were without effect on cyclic GMP levels. The hypothesis of an inhibitory effect of cyclic GMP on neutrophil function was further supported by the attenuation of SIN-1-induced inhibition of enzyme release by methylene blue (10 μM), an inhibitor of soluble guanylate cyclase. Moreover, 8-bromo cyclic GMP and dibutyryl cyclic GMP, 1 mM, decreased ß-glucuronidase release from FMLP-stimulated PMN by 12 and 44% of the control, respectively. These data demonstrate that cyclic GMP is an inhibitory second messenger in human PMN and suggest that this action of SIN-1 may be of considerable interest under conditions of platelet/PMN activation, e.g. during myocardial ischemia.

Effects of nicotine on the functions of human polymorphonuclear leukocytes in vitro.

Effects of nicotine on migration, extracellular release of lysosomal enzymes, and superoxide anion (O-2) production of human polymorphonuclear leukocytes (PMN) were studied. Nicotine (5 X 10(-6) to 5 X 10(-4) M) had no effect on random migration, chemotaxis to fMet-Leu-Phe, nor on chemokinesis induced by fMet-Leu-Phe. Nicotine, however, inhibited both extracellular release of lysosomal enzymes from PMN and O-2 production of PMN, both of which were induced by fMet-Leu-Phe and cytochalasin B. The inhibition of enzyme release and O-2 production by nicotine was not affected by atropine, hexamethonium, or acetyl beta-methylcholine, suggesting a direct action of nicotine on PMN functions. It is presumed that nicotine does not affect PMN migration to inflammatory sites, but inhibits the microbicidal functions of PMN. Exposure to PMN to nicotine introduced into the body by smoking could suppress their functions. This might result in harmful influences on the host defense mechanism, including antitumor function.

Effects of various anti-inflammatory and anti-rheumatic agents on the synthesis, secretion, and activity of a cartilage proteoglycan-degrading enzyme and other macrophage enzymes

Enzymes released from the macrophage have been implicated in the connective tissue loss noted in a number of chronic inflammatory diseases. The effects of 2- and 3-day exposures to glucocorticosteroids, non-steroidal anti-inflammatory drugs and selected anti-rheumatic agents on release of a cartilage proteoglycan-degrading enzyme, and of β-glucuronidase, cathepsin D, and elastase from the thioglycollate-elicited mouse peritoneal macrophage were determined. The synthesis (and/or release) of the cartilage proteoglycan degrading enzyme was markedly inhibited following exposure to dexamethasone, flufenamic acid, chloroquine and gold sodium thiomalate. It was unaffected by exposure to aspirin, naproxen, indomethacin and dl-penicillamine, but was and stimulated by levamisole. Dexamethasone stimulated β-glucuronidase synthesis and release and inhibited elastase synthesis. The synthesis and release of β-glucuronidase, cathepsin D and elastase were not affected by non-toxic concentrations of indomethacin and chloroquine. These results demonstrate that certain anti-rheumatic and anti-inflammatory agents may derive at least a part of their therapeutic effects from the inhibition of enzyme release from the macrophage.

The Induction of Lysosomal Enzyme Release from Leucocytes of Normal and Emphysematous Subjects and the Effects of Tobacco Smoke upon Phagocytosis

1. The lysosomal enzymes of circulating polymorphonuclear leucocytes contain a potent elastase; release of this enzyme within the lung is thought to be responsible for the destruction of elastic tissue in pulmonary emphysema. 2. The release of lysosomal enzymes from blood leucocytes of normal and emphysematous subjects during phagocytosis of particulate material was studied In vitro. Acid phosphatase and acid ribonuclease were used as markers of lysosomal enzyme release, no sufficiently sensitive assay for elastase being available. Cigarette smoke was separated into ‘particulate’ and ‘soluble’ fractions. In a preliminary study, the particulate fraction stimulated enzyme release; in the experiments reported here, latex particles were used to produce this effect. 3. Approximately one-third of the total lysosomal enzyme content was released to the exterior of the cell during phagocytosis of latex particles. In this respect there was no difference between normal and emphysematous subjects. 4. The effects of the non-particulate soluble fraction of cigarette smoke on phagocytosis-induced enzyme release were studied. This fraction inhibited enzyme release from polymorphonuclear leucocytes of normal subjects but not from those of emphysematous patients. When the ‘cigarette-smoke solution’ was replaced by the respiratory inhibitor, antimycin A, a similar inhibition of enzyme release occurred. The inhibition of phagocytosis in cells of normal subjects is presumed to be due to a respiratory inhibitor such as carbon monoxide in the soluble fraction of the smoke. We postulate that the polymorphonuclear leucocytes of emphysematous patients are adapted to hypoxic conditions so that inhibition of enzyme release does not occur.

The Significance of Intracellular Calcium in Rat Liver Cell Damage by Carbon Tetrachloride

Introduction Early after intraperitoneal injection of carbon tetrachloride there is an influx of calcium into the liver cell ( Reynolds, 1963; Rees, 1962). A previous study ( Kröner, 1973) showed that increased intracellular calcium level is correlating with the loosing of cytoplasmatic enzymes. On the other hand Minot (1929) and Cantarow (1938) described a protective effect of calcium against the toxic manifestation of carbon tetrachloride poisoning. Varying the rat calcium uptake by giving vitamin D 3 or calcium gluconate we studied the influence of calcium on the release of enzymes and electrolyte shift in the early phase of liver cell injury. Material and Methods Experiments were performed with female Wistar rats, weighing 160–220 g and maintained on Altromin standard diet and water ad libitum. The rats were killed 2, 4 and 6 hours after intraperitoneal injection of carbon tetrachloride (1 ml/kg body weight). Calcium and magnesium were determined in serum and liver by atomic absorption spectrophotometry. Potassium and sodium were estimated by flame photometry. The activity of alanine-transaminase and lactate-dehydrogenase in serum was determined spectrophotometrically (Biochemica Test Combination, Boehringer Mannheim GmbH). Vitamin D 3 (Bayer/Merck) diluted in olive oil was applied by stomach tube 72 hours before experiments were started. Calcium gluconate solution was given simultaneously with carbon tetrachloride intraperitoneal. Results and Discussion The early rise of liver calcium after application of carbon tetrachloride was much larger in animals treated additionally with calcium gluconate or pretreated with vitamin D 3. In contrast there were no distinct differences in plasma calcium. The additional treatment further caused a lesser resp. later shift of sodium and potassium in liver and an inhibition of enzyme release. The effects are specific for carbon tetrachloride poisoning. Analysis of intracellular distribution of calcium led us to the assumption of two different phenomena for the biphasic increase of calcium in liver described by Reynolds (1964) in carbon tetrachloride poisoning.

Role of microtubule assembly in lysosomal enzyme secretion from human polymorphonuclear leukocytes. A reevaluation.

The dose-related inhibition by colchicine of both lysosomal enzyme release and microtubule assembly was studied in human polymorphonuclear leukocytes (PMN) exposed to the nonphagocytic stimulus, zymosan-treated serum (ZTS). Cells were pretreated with colchicine (60 min, 37 degrees C) with or without cytochalasin B (5 microng/ml, 10 min) and then stimulated with ZTS (10%). Microtubule numbers in both cytochalasin B-treated and untreated PMN were increased by stimulation and depressed below resting levels in a dose-response fashion by colchicine concentrations above 10(-7) M. These concentrations also inhibited enzyme release in a dose-response fashion although the inhibition of microtubule assembly was proportionately greater than the inhibition of enzyme release. Other aspects of PMN morphology were affected by colchicine. Cytochalasin B-treated PMN were rounded, and in thin sections the retracted plasma membrane appeared as invaginations oriented toward centrally located centrioles. Membrane invaginations were restricted to the cell periphery in cells treated with inhibitory concentrations of colchicine, and the centrioles and Golgi apparatus were displaced from their usual position. After stimulation and subsequent degranulation, the size and number of membrane invaginations greatly increased. They remained peripheral in cells pretreated with greater than 10(-7) M colchicine but were numerous in the pericentriolar region in cells treated with less than 10(-7) M. Similarly, untreated PMN that were permitted to phagocytose immune precipitates had many phagosomes adjacent to the centriole. After colchicine treatment, phagosomes were distributed randomly, without any preferential association with the centrioles. These data suggest that microtubules are involved in maintaining the internal organization of cells and the topologic relationships between organelles and the plasma membrane.

Regulation of lysosomal enzyme secretion: role in inflammation.

The purpose of this review is first to discuss the lysosomal concept as it applies to the mediation of tissue injury, and second to provide new evidence for the regulation of lysosomal enzyme secretion from human neutrophils by cyclic nucleotides, autonomic neurohormones, prostaglandins, glucocorticosteroids and calcium. Lysosomal enzymes gain access to the extracellular environment by the selective secretion of lysosome granule contents from neutrophils during cell contact with various immune reactants. Discharge of lysosomal contents results in the provocation of acute inflammation and connective tissue degradation. The immunologic secretion of lysosomal enzymes from human neutrophils requires the presence of extracellular calcium and can be modulated by several different classes of hormones, drugs and other agents. Agents that enhance lysosomal enzyme secretion also produce a concomitant accumulation of cyclic GMP (guanosine 3′, 5′-monophosphate) in neutrophils. Such substances include immune reactants, acetylcholine, prostaglandin F2α and calcium ionophores. Cyclic GMP itself also stimulates lysosomal enzyme secretion. On the other hand, inhibition of enzyme release is frequently accompanied by the accumulation of intracellular cyclic AMP (adenosine 3′, 5′-monophosphate). These effects are produced by epinephrine (catecholamines) and several prostaglandins. Cyclic AMP also inhibits lysosomal enzyme secretion. Glucocorticosteroids also inhibit enzyme secretion but this effect is accompanied by an inhibition of cyclic AMP accumulation rather than by the stimulation of cyclic AMP accumulation. The presence of cyclic GMP, cyclic AMP and the cellular processes for accumulating either nucleotide provide the human neutrophil with the means by which to modulate the secretion of lysosomal contents in response of the cells to various endogenous substances and drugs.

Hormonal control of neutrophil lysosomal enzyme release: effect of epinephrine on adenosine 3',5'-monophosphate.

Human neutrophilic leukocytes release neutral protease and beta-glucuronidase during cell contact with, and phagocytosis of, zymosan particles treated with rheumatoid arthritic serum. Release of lysosomal enzymes is inhibited by epinephrine and adenosine 3',5'-monophosphate (cyclic AMP), but not by phenylephrine or adenosine 5'-monophosphate. Inhibition of enzyme release by epinephrine may be mediated by cyclic AMP because the cyclic AMP in the neutrophils is increased by epinephrine treatment at the time when enzyme release is reduced.

The effects of low concentrations of glutaraldehyde on Micrococcus lysodeikticus membranes: Changes in the release of membrane-associated enzymes and membrane structure

The manner in which enzymes are associated with the plasma membrane of Micrococcus lysodeikticus can be modified in certain instances by the action of an aldehyde cross-linking agent. Enzymes such as ATPase (EC 3.6.1.3), NADH dehydrogenase (EC 1.6.99.3) and polynucleotide phosphorylase (EC 2.7.7.8) can normally be selectively released from the membrane by controlled washing with buffers. This release phenomenon is prevented when membranes are first treated with 0.5% (v/v) glutaraldehyde. Under these conditions the enzymes become more strongly attached to the membrane while retaining activity. In the case of NADH dehydrogenase it can be shown that the total activity is unchanged after its association with the membrane has been strengthened by this treatment. The inhibition of enzyme release is supported by the results of polyacrylamide gel electrophoresis and by the fact that glutaraldehyde treated membranes constitute a larger fraction of total cell protein than untreated cell membranes. Electron microscopy reveals that the selective release of enzymes from untreated membranes is associated with a progressive reduction in granular substructure on the membrane surface. The smooth surfaced residue which remains, contains cytochromes and succinate dehydrogenase and is resistant to further release by washing. Membranes treated with glutaraldehyde retain the granular substructure despite the washing procedures.