A hypothesis on the metabolism of glyceryl trinitrate in vascular endothelial cells.

BACKGROUND: Extensive research has been conducted regarding the mechanism of action of glyceryl trinitrate (GTN). It is currently believed that GTN undergoes a thiol-dependent metabolic pathway and releases its active metabolite, nitric oxide (NO) and/or S-nitrosothiols (R-SNO). This activates guanylyl cyclase (GC) leading to the formation of cGMP, which is responsible for the relaxation of vascular smooth muscles and the inhibition of platelet aggregation. The lack of knowledge as to the precise mechanism of GTN action and the modulation of its formation has limited the prevention of tolerance to GTN. RESULTS: With cultured human vascular endothelial cells (EC), we showed that nitrite was first formed in endothelial cells whose concentration was dependent on reduced thiols. Cells preexposed to GTN significantly decreased the production of nitrite compared with cells that were not preexposed. Furthermore, we showed that thiols in cultured cells were oxidized during interaction with GTN, which correlated with the time of exposure to GTN. CONCLUSION: Nitrite is the first active intermediate of GTN metabolism in endothelial cells. The analysis of the changes of the blood nitrite and reduced thiols concentration is helpful for evaluating the vasodilatation activity of GTN during therapeutic treatments.

Nitrite May Be an Important EDRF Modulator.

Recently many studies suggested that S-nitrosothiols instead of active nitric oxide are EDRF. The studies showed that reduced thiols could not react directly with.NO at physiological pH (7.4) to form S-nitrosothiols but oxidized to nitrite which is very stable at pH 7.4. In contrast at low pH nitrite reacted rapidly with reduced thiols to form S-nitrosothiols. Similarly both nitrite and.NO gas reacted with deoxyHb and oxyHb at low pH but not at pH 7.4 to form HbFeII-NO complex or metHb and nitrate. The pH is a key factor to control this nitrosation process which is determined in vivo by oxygen tension and redox status. From these results a hypothesis is proposed that nitrite may be an EDRF modulator which controls the nitrosation level of reduced thiols in biological system.

Anti-inflammatory activity of a lipid fraction (lyprinol) from the NZ green-lipped mussel.

A lipid-rich extract, preparared by supercritical fluid extraction of fresh stabilized mussel powder (Lyprinol), showed significant anti-inflammatory (AI) activity given therapeutically and prophylactically po to Wistar and Dark Agouti rats developing either (a) adjuvant-induced polyarthritis or (b) collagen(II)-induced autoallergic arthritis, with ED(50)/=25 mg/kg or various therapeutic oils (flaxseed, evening primrose, fish)>/=1800 mg/kg given orally. Lyprinol showed little or no activity in acute irritation assays (carrageenan, kaolin, histamine) indicating it is not mimicking rapid-acting NSAIDs.Incorporating Lyprinol into arthritigenic adjuvants composed of heat-killed Mycobacterium. tuberculosis suspended in olive oil or squalane, effectively prevented arthritis development at a dose of 5 mg/rat. By contrast, 'dummy adjuvants' prepared with Mycobacterium tuberculosis and flaxseed, evening primrose or fish oils were still arthritigenic in Dark Agouti rats (doses of oil=90 mg/rat).Lyprinol subfractions inhibited leukotriene-B(4) biosynthesis by stimulated human polymorphonuclear leukocytes in vitro, and prostaglandin-E(2) production by activated human macrophages in vitro. Much of this AI activity was associated with polyunsaturated fatty acids and natural antoxidants (carotenoids, etc.).In contrast to NSAIDs, Lyprinol is non-gastrotoxic in disease-stressed rats at 300 mg/kg po and does not seem to affect platelet aggregation (human, rat). These data show Lyprinol to be a reproducible, relatively stable, source of bioactive lipids with much greater potency than plant/marine oils currently used as nutritional supplements to ameliorate signs of inflammation.

N-acetylcysteine in combination with nitroglycerin and streptokinase for the treatment of evolving acute myocardial infarction. Safety and biochemical effects.

BACKGROUND: N-acetylcysteine (NAC) has been shown to potentiate the effects of nitroglycerin (NTG) and to have antioxidant activity. This is the first study to assess the safety and effect of NAC in the treatment of evolving acute myocardial infarction (AMI). METHODS AND RESULTS: Patients with AMI received either 15 g NAC infused over 24 hours (n = 20) or no NAC (n = 7), combined with intravenous NTG and streptokinase. Peripheral venous plasma malondialdehyde (MDA), reduced (GSH) and oxidized (GSSG) glutathione concentrations, and rate of reperfusion (using continuous ST-segment analysis) were measured. Cardiac catheterization was performed between days 2 and 5. No significant adverse events occurred. Less oxidative stress occurred in patients treated with NAC than in patients not receiving NAC (GSH to GSSG ratio 44 +/- 25 versus 19 +/- 13 at 4 hours, P < .05). NAC concentration (mean 172 +/- 79 mumol/L at 4 hours) was correlated to GSH concentration (P = .006). MDA concentrations were lower (P = .001) over the first 8 hours of treatment with NAC. There was a trend toward more rapid reperfusion (median 58 minutes, 95% confidence interval [CI] 48 to 98 minutes versus median 95 minutes, 95% CI 59 to 106 minutes; P = .17) and better preservation of left ventricular function (cardiac index 3.4 +/- 0.8 versus 2.6 +/- 0.27 L.min.m2, P = .009) with NAC treatment. CONCLUSIONS: NAC in combination with NTG and streptokinase appeared to be safe for the treatment of evolving AMI and was associated with significantly less oxidative stress, a trend toward more rapid reperfusion, and better preservation of left ventricular function.

Flux of intracellular labile zinc during apoptosis (gene-directed cell death) revealed by a specific chemical probe, Zinquin.

BACKGROUND: The transition metal Zn(II) is thought to regulate cell and tissue growth by enhancing mitosis (cell proliferation) and suppressing the counterbalancing process of apoptosis (gene-directed cell death). To investigate the role of Zn(II) further, we have used a UV-excitable Zn(II)-specific fluorophore, Zinquin. The ester group of Zinquin is hydrolyzed by living cells, ensuring its intracellular retention; this allows the visualization and measurement of free or loosely-bound (labile) intracellular Zn(II) by fluorescence video image analysis or fluorimetric spectroscopy. RESULTS: Here we show that in cells undergoing early events of apoptosis, induced spontaneously or by diverse agents, there is a substantial increase in their Zinquin-detectable Zn(II). This increase occurred in the absence of exogenous Zn(II) and before changes in membrane permeability, consistent with a release of Zn(II) from intracellular stores or metalloproteins rather than enhanced uptake from the medium. We propose that there is a major redistribution of Zn(II) during the induction of apoptosis, which may influence or precipitate some of the later biochemical and morphological changes. CONCLUSIONS: The phenomenon of Zn(II) mobilization, revealed by Zinquin, presents a new element in the process of apoptosis for investigation and may permit rapid and sensitive identification of apoptotic cells, particularly in those tissues where their frequency is low.

Video image analysis of labile zinc in viable pancreatic islet cells using a specific fluorescent probe for zinc.

We used an intracellular zinc-specific fluorophore, Zinquin, in conjunction with fluorescence video image analysis, to reveal labile zinc in pancreatic islet cells, which concentrate this metal for use in synthesis, storage, and secretion of insulin. Zinquin vividly demonstrated zinc in the islet cell secretory granules, which formed a brightly labeled crescent in the cytoplasm between one side of the nucleus and the plasma membrane. Lower but still appreciable amounts of zinc were detected in the remaining cytoplasm, but there was little labeling in the nucleus. Fluorescence intensity varied among islet cells, suggesting differences in zinc content. Their average fluorescence intensity greatly surpassed that of the surrounding pancreatic acinar cells in frozen sections of pancreas and in all other types of cell studied, including lymphocytes, neutrophils, fibroblasts, and erythrocytes. Less labile zinc was detected in cells of the mouse insulinoma cell line NIT-1, regardless of whether they were maintained in long-term culture in the presence or absence of exogenous extracellular zinc. Exposure of islet or insulinoma cells to a high concentration of glucose or other secretagogue decreased the content of labile zinc. Zinquin should be a useful probe for revealing changes in zinc homeostasis in islet B-cells that may be important in their dysfunction and death during diabetes.

Correlation of apoptosis with change in intracellular labile Zn(II) using zinquin [(2-methyl-8-p-toluenesulphonamido-6-quinolyloxy)acetic acid], a new specific fluorescent probe for Zn(II).

Zinquin [(2-methyl-8-p-toluenesulphonamido-6-quinolyloxy)-acetic acid], a membrane-permeant fluorophore specific for Zn(II), was used with spectrofluorimetry and video image analysis to reveal and quantify labile intracellular Zn. Zinquin labelled human chronic-lymphocytic-leukaemia lymphocytes, rat splenocytes and thymocytes with a weak diffuse fluorescence that was quenched when intracellular Zn was chelated with NNN'N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) and was greatly intensified by pretreatment of cells with the Zn ionophore pyrithione and exogenous Zn. There was substantial heterogeneity of labile Zn among ionophore-treated cells, and fluorescence was largely extranuclear. The average contents of labile Zn in human leukaemic lymphocytes, rat splenocytes and rat thymocytes were approx. 20, 31 and 14 pmol/10(6) cells respectively. Morphological changes and internucleosomal DNA fragmentation indicated substantial apoptosis in these cells when the level of intracellular labile Zn was decreased by treatment with TPEN. Conversely, increasing labile Zn by pretreatment with Zn plus pyrithione suppressed both spontaneous DNA fragmentation and that induced by the potent apoptosis-induced agents colchicine and dexamethasone. These results suggest that prevention of apoptosis is a function of labile Zn, and that a reduction below a threshold concentration in this Zn pool induces apoptosis.

Interleukin-8 primes human neutrophils for enhanced superoxide anion production.

Interleukin-8 (IL-8), a novel chemotactic cytokine, has been shown to play an important role in inflammation. In this study, we investigated the effect of recombinant human (rh) IL-8 on superoxide (O2-) production by neutrophils. We found that rhIL-8 (1-10 ng/ml) did not stimulate neutrophil O2- production on its own, but primed neutrophils for an enhanced response to other stimuli, such as N-formyl-methionyl-leucyl-phenylalanine (FMLP), phorbol 12-myristate 13-acetate (PMA) and platelet-activating factor (PAF). The priming effect of rhIL-8 was dose dependent, rapid and long lasting. Recombinant human IL-8 increased both the maximal rate and the total O2- production, but did not prolong the response to FMLP. Stimulation of neutrophils with rhIL-8 increased intracellular-free calcium concentration ([Ca2+]i) by mobilizing calcium from internal stores and by increasing calcium influx. The increase in [Ca2+]i was dose dependent and occurred in the same range of rhIL-8 concentrations that primed neutrophils for O2- production. In addition, rhIL-8 enhanced the FMLP-stimulated increase in [Ca2+]i. These observations suggest that calcium may play an important role in priming phenomenon.

The substance P fragment SP-(7-11) increases prostaglandin E2, intracellular Ca2+ and collagenase production in bovine articular chondrocytes.

Substance P (SP) is found in increased concentrations in inflamed joints and is believed to play a role in joint pathology. Culture of bovine articular chondrocytes with SP or with the related mammalian tachykinins neurokinin A or B (NKA or NKB) produced no effect on prostaglandin E2 (PGE2) or collagenase production. However, the C-terminal fragment of SP, SP-(7-11), increased PGE2 and collagenase production at concentrations greater than 1 microM. The N-terminal fragments SP-(1-4) and SP-(1-6) had no effect on PGE2 or collagenase production. In addition, SP-(7-11), but not intact SP, SP-(1-4), SP-(1-6), SP-(8-11) or SP-(9-11), nor the tachykinins NKA and NKB, caused an increase in the intracellular calcium concentration as measured by the fluorescent dye Fura-2. The maximal change in intra-cellular calcium induced by 10 microM SP-(7-11) was 140 +/- 30 nM. We postulate that cleavage of SP by neutral endopeptidases which are present in the synovial fluid and which yield SP-(7-11) may be of biological importance in chondrocyte-mediated cartilage pathology.

Activation of human neutrophils by tachykinins: effect on formyl-methionyl-leucyl-phenylalanine- and platelet-activating factor-stimulated superoxide anion production and antibody-dependent cell-mediated cytotoxicity.

This study examines the contribution of tachykinins to the processes of inflammation. Neurokinin A (NKA), neurokinin B (NKB) and eledoisin (E) but not kassinin (K) have similar effects to substance P (SP) in priming neutrophils for increased superoxide anion (O2-) production in response to formyl-methionyl-leucyl-phenylalanine (FMLP). This similarity in activity may be due to the carboxy amino acid terminal end of these tachykinins being highly conserved. This was confirmed by demonstrating that SP fragment 7-11 (SP7-11) had the same priming effect as the whole molecule, whereas, the amino end fragment 1-4 (SP1-4) inhibited the response to FMLP. The priming effect of tachykinins was not confined to a single stimulus, such as FMLP, since NKA, NKB and SP also enhanced O2- production stimulated by platelet-activating factor (PAF), an important mediator of inflammation but a weak stimulus of O2- production on its own. In addition, all the tachykinins studied increased neutrophil antibody-dependent cell-mediated cytotoxicity (ADCC) towards opsonized target cells. In contrast to their effects on FMLP-induced O2- production, both SP fragments, SP1-4 and SP7-11, stimulated neutrophil ADCC and had a synergistic effect when used together.

Regulation of protein kinase C by Zn(2+)-dependent interaction with actin.

Zn2+ influences diverse cellular processes by poorly understood mechanisms. Some of these effects may be mediated by the protein kinase C (PKC) family of enzymes, since an influx of Zn2+ greatly increases their binding of regulatory ligand phorbol ester and induces their translocation from cytosol to the cytoskeleton. Using a model with purified components, we now show that Zn2+ acts by forming a phospholipid-dependent complex of PKC with filamentous actin, which results in expression of new binding sites for phorbol ester and phosphorylation of actin. These results provide a basis for the observed localization of PKC at actin-membrane junctions, in-vivo.

Zinc induces specific association of PKC with membrane cytoskeleton.

Putative binding sites for zinc are present in the regulatory domain of protein kinase C but a distinct role for zinc has not yet been proposed. Here we show that micromolar concentrations of zinc chloride cause pure rat brain protein kinase C to localize in a detergent-insoluble, cytoskeletal fraction of red cell membranes and to bind to isolated cytoskeleton in the presence of phosphatidylserine. Attachment of protein kinase C to cytoskeleton was accompanied by enhanced expression of binding sites for 3H-phorbol ester, a regulatory ligand of protein kinase C. The active factor in the cytoskeleton was labile to protease suggesting that protein kinase C binds to a cytoskeletal protein.

Synergy between zinc and phorbol ester in translocation of protein kinase C to cytoskeleton.

Protein kinase C was measured in the cytoskeletal fraction of lymphocytes, platelets and HL60 cells, by specific binding of [3H]phorbol dibutyrate and by immunoblotting with antibody to a consensus sequence in the regulatory domain of alpha-, beta- and gamma-isozymes of protein kinase C. Treatment of cells for 40 min with a combination of zinc (2-50 microM), zinc ionophore pyrithione and unlabelled phorbol dibutyrate (200 nM) caused up to a ten-fold increase in cytoskeletal protein kinase C and a corresponding decrease in other cellular compartments. Omission of any of the reagents resulted in much less or no translocation. These effects were inhibited by 1,10-phenanthroline, which chelates zinc, and were not seen with calcium. Increase in cytoskeletal protein kinase C persisted for several hours and appeared to involve attachment of the enzyme to actin microfilaments. We propose that zinc, like calcium, regulates the distribution of PKC in cells. However, unlike calcium which controls the binding of PKC to the lipid component on cell membranes, zinc controls the distribution of PKC to membrane cytoskeleton, possibly actin.

Auranofin stimulates LTA hydrolase and inhibits 5-lipoxygenase/LTA synthase activity of isolated human neutrophils.

The effect of auranofin on the 5-lipoxygenase pathway was studied in human neutrophils stimulated with either fMLP or A23187 (with or without arachidonic acid). The synthesis of leukotriene B4 (LTB4), 5-HETE and the all-trans isomers of LTB4 was measured by HPLC. At low concentrations (0.5-2.0 microM), auranofin stimulated LTB4 synthesis, but inhibited it at higher concentrations (100% inhibition at less than 10 microM). In contrast auranofin caused dose-dependent inhibition of the synthesis of 5-HETE and the all-trans isomers of LTB4. Similar observations were made with each agonist. The stimulation of LTB4 synthesis and inhibition of the trans isomer production suggests that auranofin at low concentrations stimulates LTA hydrolase--the enzyme that converts LTA4 to LTB4, whereas the inhibition of synthesis of all lipoxygenase products at higher auranofin concentrations, suggests inhibition of 5-lipoxygenase/LTA synthase.

Effect of dietary oils on the production of n-3 and n-6 metabolites of leukocyte 5-lipoxygenase in five rat strains.

We examined the influence of various dietary oils, including linseed and fish oil on the relative rates of leukotriene B4 (LTB4) and LTB5 production by rat peritoneal exudate cells in five rat strains. While there was an association between the membrane phospholipid levels of the fatty acid precursors (arachidonic acid (AA) and eicosapentaenoic acid (EPA)) and the rate of synthesis of their respective 5-lipoxygenase products (LTB4 and LTB5), the rate of LTB4 synthesis was a combined function of both AA and EPA levels. We observed a strong linear relationship (correlation coefficient = 0.99) between the ratio of EPA/AA in the cell membrane phospholipids and the ratio of LTB5/LTB4 produced by these cells in vitro; this association was independent of genetic (strain) variability and was independent of the source of EPA (dietary EPA or EPA endogenously synthesized from dietary alpha-linolenic acid).

Phorbol 12-myristate 13-acetate, A23187 and L-adrenaline inhibit phospholipid methylation in human monocytes and lymphocytes. Inhibition is independent of oxyradical production and phospholipid hydrolysis.

The Ca++ ionophore A23187 and phorbol 12-myristate 13-acetate (PMA) caused dose-dependent inhibition of phospholipid (PL) methylation in unfractionated mononuclear cells (MNC), monocytes, and lymphocytes as measured by incorporation of 3H-methyl-groups from [3H-methyl]-L-methionine into phosphatidylcholine (PC), dimethyl phosphatidylethanolamine (PE), and monomethyl PE. This inhibitory effect did not correlate with monocyte superoxide release and was unaltered by the presence of either catalase and superoxide dismutase or the NADPH oxidase inhibitor, diphenylene iodonium (DPI), indicating that oxyradical-mediated oxidation of methionine was not the major cause of inhibition of PL methylation. Furthermore L-adrenaline, which elevates cAMP and does not stimulate superoxide release, also inhibited PL methylation. Inhibition by PMA was not due to reduction in intracellular levels of methionine or S-adenosyl methionine. A23187 caused reduction of S-adenosyl methionine levels only at 1 microM, and had no effect at lower concentrations. Inhibition of PL methylation was shown not to be due to phospholipase A2-dependent hydrolysis of newly methylated PL. Attempts to reverse the inhibitory effect of either A23187 or PMA with the putative protein kinase inhibitors W-7 and H-7 were inconclusive. The mechanism of inhibition of PL methylation by A23187 and PMA remains unclear, but does not appear to be due to oxidation of methionine or hydrolysis of newly methylated PL.

Activation of human neutrophils by substance P: effect on FMLP-stimulated oxidative and arachidonic acid metabolism and on antibody-dependent cell-mediated cytotoxicity.

We show that the neuropeptide, substance P (SP), a putative mediator of neurogenic inflammation, is a potent regulator of mature, human neutrophil function. SP increased neutrophil cytotoxic activity against an antibody-coated target (P815 cells) in a dose-dependent manner. The maximal effect was noted at an SP concentration of 10(-4) M, when cytotoxicity increased from 4.7 +/- 0.9% to 33.4 +/- 10.3%. This effect was not due to toxicity of SP against the target cells and was antibody-dependent. The level of cytotoxic activity induced by SP was comparable to that described for a number of cytokines, such as GM-CSF, under identical assay conditions. SP-induced cytotoxicity was 73.1 +/- 5.8% of that produced by an optimum concentration of conditioned medium known to contain a number of cytokines which activate mature neutrophils. In addition, SP enhanced FMLP-stimulated superoxide anion production by neutrophils in a dose-dependent fashion. Neutrophils preincubated with medium or 7.5 x 10(-5) M SP and then stimulated with 10(-7) M FMLP produced 7.9 +/- 2.7 and 29.9 +/- 3.7 nmol superoxide anion/10(6) cells, respectively. This priming effect of SP was rapid in onset (less than 15 min) and was maximal from 15 to 60 min, after which it declined. It was not reversed by washing the cells and was temperature dependent. SP did not shift the dose-response curve to FMLP to the left, but it enhanced the response to FMLP in the concentration range 10(-8)-10(-6) M. Similarly SP enhanced LTB4 and 5-HETE production by FMLP-stimulated but not calcium ionophore-stimulated neutrophils. Therefore, these data provide evidence that SP regulates a number of neutrophil functions and suggests a mechanism whereby the nervous system may affect the immune response. Furthermore, the regulatory effects of SP on the neutrophil functions studied appear to be similar to those of a number of cytokines that have been previously implicated in inflammation.