Effect of anti-inflammatory drugs on lysosomes and lysosomal enzymes from rat liver

Steroidal and nonsteroidal anti-inflammatory drugs were tested for their capacity to stabilize, in vitro, lysosomes and inhibit lysosomal enzymes. Lysosome membrane stability was measured by determining the effects of drugs on the release of aryl sulfatase and β-glucuronidase from lysosomes which were suspended in a hypo-osmotic sucrose buffer. Lysosomes obtained from a heavy mitochondrial (3500 g) rat liver fraction were found to be highly sensitive to membrane stabilization by naproxen, alclofenac, chloroquine, mefenamic acid, phenylbutazone, hydrocortisone, dexamethasone and methylprednisolone. Ibuprofen and flufenamic acid demonstrated moderate stabilizing activity, while indo-methacin, aspirin and clonixin showed only weak activity. Imuran, as well as other anti-inflammatory drugs, was inactive. In addition to their membrane-stabilizing activity, chloroquine was found to be a potent inhibitor of aryl sulfatase and phenylbutazone an inhibitor of β-glucuronidase activity. Hydrocortisone, dexamethasone and paramethasone inhibited aryl sulfatase activity, while no steroid tested was effective as an inhibitor of β-glucuronidase. The data in this report support the hypothesis that anti-inflammatory drugs inhibit the release of enzymes from lysosomes. In addition, several of these drugs may act as inhibitors of lysosomal enzyme activity.     

Effects of mefloquine on the release of marker enzymes from the rat liver crude lysosomal fraction

The effects of the antimalarial agent mefloquine on the release of marker enzymes, acid phosphatase and beta glucuronidase, from the rat liver lysosomes in a crude lysosomal preparation were investigated and compared with that of chloroquine whose membrane effects have been well-documented in the literature. At 10 microM, mefloquine decreased significantly the release of marker enzymes when compared to the control, but at the higher concentrations of 100 microM and 500 microM, it markedly accelerated the release of enzymes. This suggested that mefloquine exerted a concentration-dependent biphasic effect of membrane stabilization and labilization. In comparison, chloroquine diminished the release of enzymes over the concentration range of 10-500 microM, that is showing a membrane stabilizing effect similar to other reports. Since serum levels of 1.6-3.2 microM are reported after a weekly dose of mefloquine, the present results suggest that a predominant stabilization effect should prevail under these conditions. However, higher drug concentrations may result in labilization with undesirable consequences.     

Di-(2-ethylhexyl) phthalate enhances the release of lysosomal enzymes from alveolar macrophages during phagocytosis

Lung tissue damage, histologically similar to protease induced lung lesions, has been previously demonstrated in animals exposed to the plasticizer, di-(2-ethylhexyl)phthalate (DEHO). In an attempt to identify the mechanism responsible for this damage, we have examined the effect of DEHP on alveolar macrophages. Serum solubilized DEHP has a significant effect on both the phogocytosis of latex particles and lysosomal enzyme released from rabbit alveolar macrophages. Pre-exposure to 2 mg% DEHP caused a 2-fold increase in the rate of phagocytosis and an 8-fold and 10-fold increase, respectively, in the release of the lysosomal hydrolases β-glucuronidase and acid phosphatase. Although exposure to 2 mg% DEHP caused an 8-fold increase in in vitro cell death, pre-exposure to DEHP had only minimal effect on death during subsequent cell culture, as indicated by measurement of dye exclusion and the release of the cytosolic enzyme lactate dehydrogenase. The relationship between the DEHP induced increase in lysosomal enzyme release from alveolar macrophages and the pathological and histological effects of DEHP on pulmonary tissue is discussed, particularly with respect to patients receiving multiple blood transfusions.     

Effects of anti-inflammatory and immuno-modulating agents on the release of beta-glucuronidase and collagenase from cultured macrophages of guinea pigs.

The effects of various drugs on zymosan-induced release of β-glucuronidase (a lysosomal enzyme) and lipopolysaccharide (LPS)-induced secretion of collagenase from cultured macrophages of guinea pigs were investigated. Dexamethasone, hydrocortisone and prednisolone inhibited the release of βglucuronidase. Dexamethasone, however, did not show such an effect, when the drug was added to macrophage culture after particle uptake had completed. In contrast, indomethacin and disodium N(carboxyphenyl) 4-chloroanthranilate (CCA) significantly enhanced the enzymic release. Aspirin and levamisole enhanced it slightly. But there were no distinct differences between the effects of these agents on LPSinduced secretion of collagenase in contrast to the case of β-glucuronidase release.     

Leucocyte migration and lysosomal enzymes release in rat carrageenin pleurisy.

The time course of rat carrageenin pleurisy has been studied. The inflammatory reaction is characterized by exudate formation and massive leucocyte emigration into the pleural space both reaching peak values at 24 hours. Moreover betaglucuronidase, acid phosphatase and lactic dehydrogenase have been assayed in the exudate. The activity of lysosomal enzymes parallels the severity of the inflammatory response, while that of cytoplasmic enzyme lactic dehydrogenase resulted unmodified. Treatment of animals with indomethacin, phenylbutazone, aspirin and flufenamic acid inhibited both exudate formation and leucocyte emigration. In contrast none of these drugs was able to reduce lysosomal enzyme release.     

Effects of the nonsteroidal anti-inflammatory compounds Lonazolac Ca, indomethacin and NDGA on inflammatory mediator generation and release from various cells.

The effects of Lonazolac calcium in modulating histamine release, leukotriene and 12-HETE generation and PAF metabolism from various cells were compared with the cyclooxygenase and lipoxygenase inhibitors indomethacin and nordihydroguaiaretic acid (NDGA). For histamine release cells were stimulated with Ca ionophore, A23187. The release of histamine from human basophils was significantly decreased after preincubation of the cells with Lonazolac Ca. Lonazolac Ca and NDGA were equally potent and their activity exceeded that of indomethacin. Preincubation of human polymorphonuclear leukocytes (PMN) with Lonazolac Ca, indomethacin and NDGA at high concentrations led to an inhibition of leukotriene generation induced by either the Ca ionophore or opsonized zymosan. Different concentrations were required for inhibiting the enzymatic and the non-enzymatically generated leukotrienes. Lonazolac Ca was more potent than indomethacin. Incubation of PMN with Lonazolac calcium modulated the metabolism of exogenously added 3H-PAF and 3H-lyso-PAF. In comparison with NDGA. Lonazolac Ca affected different enzymes of the PAF metabolism.     

Changes in cellular enzyme levels and the inhibition of selective release of lysosomal hydrolases from macrophages by indomethacin.

Indomethacin increases the cellular levels of several lysosomal enzymes in cultures of mouse peritoneal macrophages exposed to the drug for periods of time ranging from one day to four weeks. This increase can be blocked by puromycin, an inhibitor of protein synthesis. Pretreatment of macrophages with indomethacin inhibits the selective release of lysosomal enzymes induced by a C-mucopolysaccharide peptidoglycan complex purified from the cell walls of Group A streptococci.     

Effect of sodium thiomalate on immune complex-induced release of lysosomal enzymes from human polymorphonuclear leucocytes

The extracellular release of beta-glucuronidase and beta-N-acetylglucosaminidase from normal human polymorphonuclear leucocytes initiated with bovine serum albumin/anti-bovine serum albumin immune complex (15 micrograms/ml) was significantly inhibited (p less than 0.01) by pretreatment with increasing concentrations (10(-8) M, 10(-7) M, 10(-6) M and 10(-5) M) of sodium thiomalate in a time- and dose-related fashion. Also, beta-glucuronidase and beta-N-acetylglucosaminidase exhibited similar responses to the effects of bovine serum albumin/anti-bovine serum albumin or sodium thiomalate. In contrast, neither bovine serum albumin/anti-bovine serum albumin nor sodium thiomalate provoked appreciable leakage of the cytoplasmic enzyme, lactate dehydrogenase. This indicates that cell integrity remains intact under the experimental conditions described.     

A comparison of anti-inflammatory and other compounds on the spontaneously contracting pregnant rat uterus.

The use of tissue from the 17 to 21 day pregnant rat uterus was based on earlier experiments showing that locally produced prostaglandins are the mediators of the spontaneous contractions in the pregnant rat uterus. The isolated uterine tissue provides a simpler method for screening compounds for inhibition of prostaglandin synthetase activity than the biochemical assays or the rat-paw-edema technique. This preparation was studied as a screening method for new antiinflammatory compounds. A 4-5 cm length of tissue from the uterus of 17-21 day pregnant Fullinsdorf rats was suspended in Kreb's bicarbonate solution and gassed with 5% carbon dioxide in oxygen. After 30 minutes to equilibrate, regular spontaneous contractions were recorded up to 8 hours. The sensitivity of this preparation to antiinflammatory agents was 10-100 times lower than reported by some others. Substances tested included papaverine, ketamine, isoprenaline, chloropromazine, desipramine, diazepam, and procaine. Only chlorpromazine had antiinflammatory activity as indicated by the ability to prevent carageenan-induced edema of the rat paw. Low concentrations of papaverine, ketamine, and isoprenaline initially reduced the force but increased the rate of contractions while chlorpromazine, desipramine, and diazepam decreased the force without affecting the rate of contraction. The known potent antiinflammatory agents, iodomethacin, phenylbutazone, acetylsalicyclic acid, and flufenamic acid reduced both rate and force of contractions of the preparation. Contractions of the preparations were completely inhibited by high doses of all these compounds. Results suggest that although isolated pregnant rat uterine tissue is a simple technique for detecting compounds that inhibit prostaglandin synthetase activity, there is lack of specificity of the preparation. Some compounds, e.g., indomethacin, have other independent effects on smooth muscle tissue.     

Phagocytic release of lysosomal enzymes from guinea pig neutrophils--regulation by corticosteroids, autonomic neurohormones and cyclic nucleotides.

The effects of various pharmacologic agents on the capacity of guinea pig neutrophils to phagocytize serum-treated zymosan particles and release lysosomal enzymes were determined. Neutrophils (10⁷) and zymosan were incubated in Krebs-Ringer phosphate (KRP) medium containing 7.5 mM glucose, pH 7.4, at 37° in the absence and presence of corticosteroids, cyclic nucleotides, and adrenomimetic and cholinomimetic agents. Methylprednisolone hemisuccinate, triamcinolone acetonide, dexamethasone acetate, paramethasone acetate and hydrocortisone hemisuccinate reduced particle uptake by and discharge of lysosomal enzymes from guinea pig neutrophils. Aldosterone hemisuccinate and deoxycorticosterone acetate were inactive. Adrenomimetic (e.g. epinephrine) agents inhibited particle uptake by and lysosomal enzyme secretion from neutrophils, and cholinomimetic (e.g. acetylcholine) agents accelerated lysosomal enzyme release but had no effect on phagocytosis. Cyclic 3',5'-adenosine monophosphate (cyclic AMP) and one of its analogs inhibited particle ingestion by and lysosomal enzyme release from neutrophils; and this inhibition was potentiated by theophylline. Cyclic 3',5'-guanosine monophosphate (cyclic GMP), in contrast to the actions of corticosteroids, adrenomimetic agents and cyclic AMP, accelerated lysosomal enzyme secretion but had no effect on particle uptake. Cyclic GMP did not affect release of cytoplasmic lactate dehydrogenase, thus indicating maintenance of cell viability during release of lysosomal enzymes. Cytochalasin B, an agent which blocked phagocytic uptake of zymosan, did not interfere with inhibition of lysosomal enzyme secretion by corticosteroids, adrenomimetic agents and cyclic AMP or acceleration of this event by cholinomimetic agents or cyclic GMP. These studies indicate that guinea pig neutrophils are capable of releasing lysosomal enzymes during phagocytosis of zymosan and that certain agents can modulate lysosomal enzyme secretion and/or phagocytosis.     

Effects of inflammatory agents on endothelial lysosomal fragility and their inhibition by anti-inflammatory drugs.

1. Endothelial cells from human umbilical veins were maintained in tissue culture. The fragility of lysosomal membranes were studied by microdensitometry. 2. Histamine (50 microM to 10 mM), 4-methylhistamine (100 nM to 10 mM) and dimaprit (100 nM to 10 mM) increased lysosomal fragility. 2-Thiazolylethylamine and 2-pyridylethylamine (100 nM to 10 mM) had no effect. 3. Prostaglandins E1 and E2 (3 nM to 30 microM) and prostaglandin F2 alpha (2 nM to 20 microM) had no direct effect. Low concentrations of prostaglandins E1 and E2 inhibited the fragility induced by histamine 100 microM. 4. Bradykinin (100 nM to 100 microM) decreased fragility. 5. The increase in fragility induced by histamine 100 microM or dimaprit 100 microM was inhibited by cimetidine (100 microM to 1 mM) but not by mepyramine (1 microM to 1 mM). 6. Pretreatment with indomethacin, hydrocortisone, ibuprofen and sodium salicylate caused a dose-dependent inhibition of histamine-induced fragility. Threshold concentrations were 1 pM, 100 pM, 10 nM and 10 microM, respectively. 7. Lignocaine (1 microM to 1 mM) had no direct effect and did not decrease histamine-induced fragility.     

Effect of cyclic adenosine monophosphate, 5'-(N-ethylcarboxyamido)-adenosine and methylxanthines on the release of thromboxane and lysosomal enzymes from human alveolar macrophages and peripheral blood monocytes in vitro.

We have investigated the effect of the manipulation of intracellular cyclic adenosine monophosphate (cyclic AMP) and the stimulation of adenosine receptors on the function of human alveolar macrophages in vitro. Human alveolar macrophages harvested by bronchoalveolar lavage were stimulated by opsonised zymosan 1 mg/ml in the presence of N6,2'0-dibutyryladenosine 3':5' cyclic monophosphate (dibutyryl cyclic AMP) 5 x 10(-6) to 5 x 10(-3) M,8-bromoadenosine 3':5'-cyclic monophosphate (8-bromo cyclic AMP) 5 x 10(-6) to 5 x 10(-3) M, 5'-(N-ethylcarboxamido)-adenosine (NECA) 10(-7) to 10(-4) M, adenosine 10(-7) to 10(-4) M, theophylline 5 x 10(-6) to 5 x 10(-3) M and enprofylline 5 x 10(-8) to 5 x 10(-4) M. The subsequent release of thromboxane B2 (TXB2) and N-acetyl-beta-D-glucosaminidase (NAG) activity was monitored. In addition, the release of TXB2 and NAG from zymosan stimulated human monocytes incubated in the presence of NECA 10(-7) to 10(-4) M was measured. The TXB2 release from alveolar macrophages were inhibited by dibutyryl cyclic AMP and 8-bromo cyclic AMP and to a lesser extent by NECA, theophylline and enprofylline. However, adenosine had no effect. None of the agents studied altered NAG release. In addition, monocytes showed greater sensitivity to the inhibitory effects of 5-N-ethylcarboxamido adenosine than alveolar macrophages. In conclusion, the alveolar macrophage was inhibited by stable analogues of cyclic AMP and xanthines at supratherapeutic concentrations but have no functional excitatory adenosine receptors and only a residual inhibitory adenosine receptor function compared to the precursor monocyte.     

Prostaglandin release from macrophages: an assay system for anti-inflammatory drugs in vitro

It is the intention of this communication to present a simple and reliable in vitro system for the evaluation of anti-inflammatory drugs. The system described appears to combine the advantages of two established procedures, i.e. measurement of the inhibition of prostaglandin (PG) synthesis in vitro using microsomal enzyme preparations and the measurement of the inhibitory effects of drugs on PG synthesis in vivo avoiding major limitations. We propose to measure the effect of drugs on the PG release from mouse peritoneal macrophages in vitro. The influence of culture conditions, phagocytosis and drugs on the PG release from these cells is described. We found that antibody-coated erythrocytes are especially suitable triggers of PG release, and that the release can be inhibited by steroidal and non-steroidal anti-inflammatory drugs in concentrations resembling their effective doses in vivo.     

The effect of non-steroidal anti-inflammatory drugs on the accumulation and release of interleukin-1-like activity by peritoneal macrophages from the mouse.

1. The non-steroidal anti-inflammatory drugs (NSAIDs) indomethacin, 10 and 100 microM, piroxicam, 100 microM, and sodium meclofenamate, 1 and 100 microM, potentiated the lipopolysaccharide (LPS)-stimulated release of interleukin-1 (IL-1)-like activity from mouse peritoneal macrophages. Aspirin up to 100 microM was without effect. The drugs did not themselves stimulate the release of IL-1-like activity at the concentrations used. 2. LPS, 1 microgram ml-1, stimulated prostaglandin E2 production by mouse peritoneal macrophages and this was totally inhibited by aspirin, 100 microM, indomethacin, 1 microM, piroxicam, 10 microM and sodium meclofenamate, 0.1 microM. 3. The potentiation of LPS-stimulated release of IL-1-like activity produced by indomethacin, 100 microM, piroxicam, 100 microM, or sodium meclofenamate, 10 microM, was inhibited by prostaglandin E2, (PGE2) 10 ng ml-1. 4. Aspirin, 100 microM, indomethacin, 100 nM to 10 microM, piroxicam, 1 to 100 microM, and sodium meclofenamate, 10 nM, all potentiated cell-associated IL-1-like activity in LPS- stimulated macrophages. The drugs had no effect on cell-associated IL-1-like activity by themselves. 5. Exogenous PGE2, 2 to 30 ng ml-1, inhibited the cell-accumulation of IL-1-like activity stimulated by LPS in the presence of indomethacin, 1 microM, or sodium meclofenamate, 0.1 microM. 6. The 5-lipoxygenase inhibitors BWA4C, 0.01 to 10 microM, and L-651,392, 0.01 to 10 microM, had no effect on LPS-stimulated released or cell-associated IL-1-like activity.(ABSTRACT TRUNCATED AT 250 WORDS)