Inhibition of lactate dehydrogenase in cultured SIRC cells by cigarette smoke

Summary SIRC cell monolayer cultures were exposed to whole smoke from a mid tar and nicotine level research cigarette (ASFC, 72 puffs), or from a high tar and nicotine level reference cigarette (Kentucky 2R1, 48 puffs) over a period of 65 days. The activity and distribution of lactate dehydrogenase (LDH) in the cells were investigated, and the electrophoretic characteristics of its isozymes studied. Cell morphology was examined by light microscopy and by transmission- and scanning electron microscopy.LDH activity was reduced by exposure to smoke from both cigarette types, the greater inhibitory effect being produced by that of the Kentucky cigarette. In addition, cells exposed to this high tar and nicotine smoke displayed intramitochondrial granules which were larger and more numerous than those found in cells exposed to the mid tar and nicotine smoke, or in the control cells. It is speculated that cation accumulation in the mitochondria may be involved in the observed inhibition of LDH activity.Supported by a research grant from the ASFC (Association Suisse des Fabricants de Cigarettes), Switzerland     

Inhibition of chloride secretion in human bronchial epithelial cells by cigarette smoke extract

Chronic bronchitis, a disease mainly of cigarette smokers, shares many clinical features with cystic fibrosis, a disease of altered ion transport, suggesting that the negative effects of cigarette smoke on mucociliary clearance may be mediated through alterations in ion transport. We tested the hypothesis that cigarette smoke extract would inhibit chloride secretion in human bronchial epithelial cells. In agreement with studies in canine trachea, cigarette smoke extract inhibited net chloride secretion without affecting sodium transport. We performed microelectrode impalements and impedance analysis studies to investigate the physiological mechanisms of this inhibition. These data demonstrated that cigarette smoke extract caused an acute increase in membrane resistances in conjunction with apical membrane hyperpolarization, an effect consistent with inhibition of an apical membrane anion conductance. After this acute phase, both membrane resistances decreased while membrane potentials continued to hyperpolarize, indicating that cigarette smoke extract also inhibited the basolateral entry of chloride into the cell. Furthermore, cigarette smoke extract caused an increase in mucin secretion. Therefore, the ion transport phenotype of human bronchial epithelial cells exposed to cigarette smoke extract is similar to that of cystic fibrosis epithelia in which there is sodium absorption out of proportion to chloride secretion in the setting of increased mucus secretion.     

Alzheimer-specific epitopes of tau represent lipid peroxidation-induced conformations

Several recent studies support a link between tau protein phosphorylation and adduction of tau by reactive carbonyls. Indeed, the phosphorylation-dependent adduction of tau by carbonyl products resulting from lipid peroxidation creates the neurofibrillary tangle-related antigen, Alz50. To determine whether epitopes of carbonyl-modified tau are major conformational changes associated with neurofibrillary tangle formation, we examined seven distinct antibodies raised against neurofibrillary tangles that recognize unique epitopes of tau in Alzheimer disease. Consistently, all seven antibodies recognize tau more strongly (4- to 34-fold) after treatment of normal tau with the reactive carbonyl, 4-hydroxy-2-nonenal (HNE), but only when tau is in the phosphorylated state. These findings not only support the idea that oxidative stress is involved in neurofibrillary tangle formation occurring in brains of Alzheimer disease patients, but also show, for the first time, that HNE modifications of tau promote and contribute to the generation of the major conformational properties defining neurofibrillary tangles.     

Inhibition of neurotransmitter release by peptides that mimic the N-terminal domain of SNAP-25

Botulinum neurotoxin serotypes A and E (BoNT/A and BoNT/E) block neurotransmitter release by cleaving the 206-amino-acid SNARE protein, SNAP-25. For each BoNT serotype, cleavage of SNAP-25 results in the loss of intact protein, the production of an N-terminal truncated protein, and the generation of a small C-terminal peptide. Peptides that mimic the C-terminal fragments of SNAP-25 following BoNT/A or BoNT/E cleavage were shown to depress transmitter release in bovine chromaffin cells and in Aplysia buccal ganglion cells. Similarly, the N-terminal–truncated SNAP-25 resulting from BoNT/A or BoNT/E cleavage has been found to inhibit transmitter exocytosis in various systems. With one exception, however, the inhibitory action of truncated SNAP-25 has not been demonstrated at a well-defined cholinergic synapse. The goal of the current study was to determine the level of inhibition of neurotransmitter release by N-terminal BoNT/A- or BoNT/E-truncated SNAP-25 in two different neuronal systems: cholinergically coupled Aplysia neurons and rat hippocampal cell cultures. Both truncated SNAP-25 products inhibited depolarization-dependent glutamate release from hippocampal cultures and depressed synaptic transmission in Aplysia buccal ganglion cells. These results suggest that truncated SNAP-25 can compete with endogenous SNAP-25 for binding with other SNARE proteins involved in transmitter release, thus inhibiting neurotransmitter exocytosis.     

Inhibition of oral peroxidase activity by cigarette smoke: in vivo and in vitro studies

Oral peroxidase (OPO), the pivotal enzyme in the salivary antioxidant system, seems to be of paramount importance in the oral defense mechanism, especially against the attack of free radicals related to cigarette smoke (CS) and the evolution of oral cancer. The major inducer of oral cancer is exposure to tobacco, which is responsible for 50-90% of cases worldwide. The purpose of our study was to elucidate the outcome of interaction between CS and OPO in smokers and nonsmokers. After smoking a single cigarette, a sharp drop of OPO activity was observed in both groups: 42.5% in smokers and 58.5% in nonsmokers (p <.05). After 30 min, the level of activity returned to 90-100% of the presmoking level, presumably due to the secretion of new saliva into the oral cavity. The difference between the two groups was also observed after exposure of saliva to one cigarette in smoking flasks (in vitro studies); however, as expected, no recovery of activity was observed in either group. Similarly, the OPO activity loss was accompanied by increased carbonylation of the salivary proteins, an indicator of the oxidative damage to proteins. These results may be of great clinical importance, as heavy smokers smoke 20 cigarettes or more on a daily basis. Accordingly, most of the time the oral epithelium of heavy smokers is essentially unprotected by OPO against the deleterious effects of thiocyanate ions and hydroxyl radicals produced by unremoved hydrogen peroxide in the presence of the salivary redox-active metal ions. This may pave the way for the CS-induced and saliva-mediated initiation and progression of oral cancer.     

Identification of glutathione modifications by cigarette smoke

Although it has been recognized for decades that cigarette smoke (CS) is toxic to respiratory tract tissues, and that glutathione (GSH) and other thiols are able to ameliorate some of the adverse effects of CS, the precise interactions between thiols and critical CS components are only partially characterized. In the present study, we used HPLC and MALDI-MS approaches to more rigorously characterize the products of CS reactions with GSH, the major cellular thiol and an important antioxidant constituent in respiratory tract lining fluids, in an attempt to increase our understanding of mechanisms of CS respiratory tract toxicity. Exposure of solutions of GSH to gas phase CS resulted in its rapid depletion, and about 50% of this depletion could be accounted for by reaction with acrolein and crotonaldehyde, the two major alpha, beta-unsaturated aldehydes in CS. Similar aldehyde adducts with GSH could also be detected in cells exposed to CS, although the relative yields were limited, presumably because of further reactions of these adducts and/or their excretion. Further characterization of in vivo thiol-aldehyde formation in respiratory tract cells can be expected to provide significant insights into the mechanisms of CS toxicity.     

Macrophage phagocytosis of apoptotic neutrophils is compromised by matrix proteins modified by cigarette smoke and lipid peroxidation products

Clearance of apoptotic cells by phagocytosis plays an important role in the resolution of an inflammatory response. Macrophages interacting with extracellular matrix (ECM) proteins upregulate their phagocytic capacity. Cigarette smoke contains highly reactive carbonyls that modify proteins which directly/indirectly affects cellular function. We observed, in vitro, that human macrophages interacting with carbonyl or cigarette smoke modified ECM proteins dramatically down regulated their ability to phagocytose apoptotic neutrophils. We also show that this interaction with carbonyl-adduct modified ECM proteins led to increased macrophage adhesion in vitro. We hypothesise that changes in the ECM environment as a result of cigarette smoking affect the ability of macrophages to remove apoptotic cells. Moreover, we postulate that this decreased phagocytic activity was as a result of sequestration of receptors involved in the uptake of apoptotic cells towards that of recognition of carbonyl adducts on the modified ECM proteins leading to increased macrophage adhesion.     

Hemolysis of rabbit erythrocytes induced by cigarette smoke

Cigarette smoke has been found to induce the hemolysis of rabbit erythrocytes. The particulate phase had more profound effect than the gas phase. Neither free radical scavengers such as ascorbic acid, uric acid and water-soluble vitamin E analogue nor antioxidant enzymes such as catalase and superoxide dismutase suppressed the cigarette smoke-induced hemolysis, suggesting that free radicals, hydrogen peroxide, and superoxide were not the active species.     

Effect of cigarette smoke on lipid peroxidation and antioxidant enzymes in albino rat

Effect of cigarette smoke on lipid peroxidation (LPX) and antioxidant enzymes like catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and glutathione-S-transferase (GST) in various organs like brain, heart, lung, liver and kidney of the albino rats exposed to cigarette smoke for 30 min/day for a period of 30 days were assayed. It was observed that the lipid peroxide levels in liver, lung and kidney were enhanced in case of animals exposed to cigarette smoke, whereas brain and heart did not show any change as compared to control animals. The activity of the antioxidant enzymes was also elevated in liver, lung and kidney of the test animals whereas, brain and heart did not show any change in the activities of all of these antioxidant enzymes except glutathione-s-transferase which was increased in brain also. The level of reduced glutathione (GSH) was lowered in liver, lung and kidney of the tested animals when compared with the control animals but there was no significant change in brain and heart. The results of our study suggest that cigarette smoke induces lipid peroxidation in liver, lung and kidney, and the antioxidant enzymes levels were enhanced in order to protect these tissues against the deleterious effect of the oxygen derived free radicals. The depletion of reduced glutathione in these organs could be due to it's utilization by the tissues to mop off the free radicals.     

Inhibition of neurotransmitter release in the lamprey reticulospinal synapse by antibody-mediated disruption of SNAP-25 function

Exocytosis - syntaxin - synaptobrevin - SNARE synaptic vesicle The lamprey giant reticulospinal synapse can be used to manipulate the molecular machinery of synaptic vesicle exocytosis by presynaptic microinjection. Here we test the effect of disrupting the function of the SNARE protein SNAP-25. Polyclonal SNAP-25 antibodies were shown in an in vitro assay to inhibit the binding between syntaxin and SNAP-25. When microinjected presynaptically, these antibodies produced a potent inhibition of the synaptic response. Ba2+ spikes recorded in the presynaptic axon were not altered, indicating that the effect was not due to a reduced presynaptic Ca2+ entry. Electron microscopic analysis showed that synaptic vesicle clusters had a similar organization in synapses of antibody-injected axons as in control axons, and the number of synaptic vesicles in apparent contact with the presynaptic plasma membrane was also similar. Clathrin-coated pits, which normally occur at the plasma membrane around stimulated synapses, were not detected after injection of SNAP-25 antibodies, consistent with a blockade of vesicle cycling. Thus, SNAP-25 antibodies, which disrupt the interaction with syntaxin, inhibit neurotransmitter release without affecting the number of synaptic vesicles at the plasma membrane. These results provide further support to the view that the formation of SNARE complexes is critical for membrane fusion, but not for the targeting of synaptic vesicles to the presynaptic membrane.     

Metals in cigarette smoke

Metals are vital for a huge number of physiological processes in the human body, but can also destroy health when the concentration is not within the physiologically favourable range. Cigarette smoking interferes with the carefully controlled metal homeostasis of the human body. This review focuses on the consequences of metal delivery to the human body by cigarette smoking and discusses the body's responses. The metal content of tobacco plants, smoke, the circulation, and various organs is discussed. Finally, we link individual cigarette smoke contained metals to the genesis of human diseases.     

Endothelial activation and injury by cigarette smoke exposure

Endothelial activation/injury following exposure to cigarette smoke may explain incidence of atherosclerosis and cardiovascular disease in smokers. We investigated cigarette smoke extract (CSE) effects relative to activation, injury, and survival of human umbilical vein endothelial cells (HUVEC) and compared circulating levels of specific endothelial activation markers between smokers and healthy non-smokers before and after smoking cessation. Viability and toxicity of HUVEC were tested by MTT and LDH assay. Release (by endothelial cells) and circulating levels (in smokers) of von Willebrand Factor (vWF), thrombomodulin (TM), was evaluated by ELISA. Incubation with increasing concentrations of CSE reduced the percentage of viable cells, being 33.9%, 23.9% after CSE 4%, 6% respectively. Dose- and time-dependent release of LDH was observed after incubation with CSE. vWF, TM release were assayed after CSE 2% HUVEC stimulation. Significant 42%, 61%, 76% increase in vWF concentration was detected respectively at 30', 60', 120'. Reduction in circulating levels of vWF, from a median value of 144.0% to 123.7%, was observed in the quitters group after smoking cessation. Exposure to cigarette smoke is cytotoxic and induces activation/injury of endothelium in vitro and in vivo. These findings may provide pathogenetic basis by which smoking can predispose to development of atherothrombosis and cardiovascular disease.     

Chemiluminescence of cigarette smoke.

Chemiluminescence from cigarette smoke (aerosol) and smoke "extracts" (suspensoids) are described. The emissons from aqueous and organic suspensoids persist for hours, are proportional to oxygen solubilities, possess energy of at least 1.8 electron volts, and display characteristics which suggest that the emissions may be partially sensitized by singlet oxygen.     

Inhibition of lecithin:Cholesterol acyltransferase activity in human blood plasma by cigarette smoke extract and reactive aldehydes

Cigarette smoking is a risk factor for atherosclerosis. It is conceivable that reactive chemical components in cigarette smoke may adversely affect reverse cholesterol transport at the level of lecithin:cholesterol acyltransferase (LCAT) and promote atherogenesis. Hence, the effect of cigarette smoke extract (CSE) on the activity of LCAT in human plasma was studied. When incubated with plasma, CSE caused both concentration- and time-dependent losses of LCAT activity. Addition of glutathione, but not ascorbate, to plasma prevented loss of LCAT activity caused by CSE. Incubation of plasma with some reactive aldehydes known to be present in cigarette smoke also inhibited LCAT activity. Among five aldehydes tested, acrolein was the strongest inhibitor of LCAT, with complete enzyme inhibition occurring at 1 mM. Acetaldehyde was the weakest inhibitor of LCAT, with 85% enzyme inhibition at 50 mM. Hexanal, formaldehyde, and malondialdehyde completely inhibited LCAT activity at 10, 50, and 50 mM, respectively. When plasma was incubated with 1 mM acrolein in the presence of 2.5 mM glutathione or dihydrolipoic acid, 100 and 57% of LCAT activity, respectively, remained after incubation. This finding suggests that reactive aldehydes may form adducts with certain free sulfhydryl groups functioning in the active site of LCAT to inhibit enzyme activity. It is concluded that reactive aldehydes are at least partially responsible for the reduction in LCAT activity in plasma treated with CSE.     

Regulation of neurotransmitter release by metabotropic glutamate receptors

The G protein-coupled metabotropic glutamate (mGlu) receptors are differentially localized at various synapses throughout the brain. Depending on the receptor subtype, they appear to be localized at presynaptic and/or postsynaptic sites, including glial as well as neuronal elements. The heterogeneous distribution of these receptors on glutamate and nonglutamate neurons/cells thus allows modulation of synaptic transmission by a number of different mechanisms. Electrophysiological studies have demonstrated that the activation of mGlu receptors can modulate the activity of Ca(2+) or K(+) channels, or interfere with release processes downstream of Ca(2+) entry, and consequently regulate neuronal synaptic activity. Such changes evoked by mGlu receptors can ultimately regulate transmitter release at both glutamatergic and nonglutamatergic synapses. Increasing neurochemical evidence has emerged, obtained from in vitro and in vivo studies, showing modulation of the release of a variety of transmitters by mGlu receptors. This review addresses the neurochemical evidence for mGlu receptor-mediated regulation of neurotransmitters, such as excitatory and inhibitory amino acids, monoamines, and neuropeptides.