Cigarette Smoke-Induced Oxidative Stress and Autophagy in Human Alveolar Epithelial Cell Line (A549 Cells).

Chronic exposure to cigarette smoke (CS) causes structural and functional changes in the respiratory tract. It is a major risk factor for cardiovascular and systemic pulmonary diseases. The aim of this study was to investigate the effect of acute CS exposure (2 h) on oxidative stress, heat shock protein 70 (HSP70) expression, autophagy (LC3 expression), and oxidative stress (DCF fluorescence) in human alveolar epithelial cell line A549. Cell culture medium was conditioned with CS using commercial cigarettes, and A549 cells were grown in modified media for 2 h. In some experiments, A549 cells were pretreated with 100 µM of L-buthionine-sulfoximine (BSO) for 24 h to induce glutathione (GSH) depletion. In the cells grown in CS-conditioned medium, GSH was depleted by more than 30%, and reactive oxygen species were increased. Moreover, there was a considerable overexpression of HSP70 and a substantial accumulation of LC3. Similar changes were found when the cells were pretreated with BSO. We conclude that the short-term exposure of epithelial cells to CS increases oxidative stress that entails enhanced autophagy activity.

Phagocytosis and Autophagy in THP-1 Cells Exposed to Urban Dust: Possible Role of LC3-Associated Phagocytosis and Canonical Autophagy.

Exposure to ambient particulate matter (PM) increases mortality and morbidity due to respiratory and cardiovascular diseases. The aim of this study was to assess the effect of standardized urban dust (UD) on phagocytosis and autophagy in a monocyte-macrophage cell line (THP-1 cells). The cells were grown for 24 h in the medium supplemented with 200 µg·mL-1 coarse carbon black (CB) or UD. In some experiments glutathione (GSH) was depleted in THP-1 cells by buthionine sulfoximine. The cells were double stained with green latex beads (phagocytosis) and with red autophagy marker (LC3) and were evaluated in a flow cytometer. In naïve THP-1 cells, about 61% of them were classified as "negative", while 39% were classified as "double-positive". Both GSH depletion and UD treatment produced three distinct subpopulations of cells on bivariate scatterplots. A new subpopulation of cells (about 24% of the total number) appeared, with a lower autophagy and phagocytosis, but with a higher autophagy/phagocytosis ratio, when compared to highly positive cells. CB affected, to some extent, phagocytosis without a substantial effect on autophagy. In conclusion, the research on distinct pathways of immune cell activation may be relevant to the diagnostics and therapy of PM-induced pneumotoxicity, inflammation, and tumorigenesis.

Urethane, but not pentobarbitone, attenuates presynaptic receptor function in rats: a contribution to the choice of anaesthetic.

BACKGROUND AND PURPOSE: We examined whether cannabinoid CB(1) and histamine H(3) receptors resemble alpha(2)-adrenoceptors in that their presynaptically mediated cardiovascular effects are less marked in urethane- than in pentobarbitone-anaesthetized pithed rats. EXPERIMENTAL APPROACH: Effects of the cannabinoid agonist CP-55,940 and the H(3) receptor agonist imetit on electrically induced tachycardic and vasopressor responses, respectively, was compared in pithed rats anaesthetized with urethane or pentobarbitone. The affinity of urethane for the three receptors was measured by radioligand binding studies in rat brain cortex membranes and its potency assessed in superfused mouse tissues preincubated with (3)H-noradrenaline. KEY RESULTS: The neurogenic tachycardic response was less markedly inhibited by CP-55,940 in urethane- than in pentobarbitone-anaesthetized pithed rats. Imetit inhibited the neurogenic vasopressor response after pentobarbitone but not after urethane. The catecholamine-induced tachycardic and vasopressor response did not differ between rats anaesthetized with either compound. Urethane 10 mM (plasma concentration reached under anaesthesia) did not affect binding to CB(1) or H(3) receptors and alpha(2) adrenoceptors, nor did it alter the inhibitory effect of agonists at the three receptors on electrically evoked (3)H-noradrenaline release. CONCLUSIONS AND IMPLICATIONS: Urethane, but not pentobarbitone, abolished the H(3) receptor-mediated vascular response in pithed rats and attenuated the CB(1) receptor-mediated cardiac response much more than pentobarbitone. The weaker effects of CB(1), H(3) and alpha(2) receptor agonists cannot be explained by antagonism by urethane at the three receptors in vitro. Pentobarbitone, but not urethane, is suitable as an anaesthetic for investigations of inhibitory presynaptic receptor function in pithed and anaesthetized rats.

Four close bupranolol analogues are antagonists at the low-affinity state of beta1-adrenoceptors.

Bupranolol is an antagonist at the cardiostimulatory low-affinity state of b(1)-adrenoceptors and we were interested whether this effect is shared by its fluorine (GD-6), methyl (DZ-51) and isopropyl analogue (DZ-13) and by the analogue hydroxylated at the tertiary butyl moiety (DZ-52). (-)-Bupranolol and compounds (-)-GD-6, (+)-GD-6, (-)-DZ-13, (+)-DZ-13, DZ-51 and DZ-52 antagonized the CGP 12177-induced tachycardia in pithed rats with "apparent pA(2) values" of 6.1, 6.1, 4.6, 5.5, 4.6, 5.1 and 5.3, respectively. For comparison, their potencies and affinities at the high-affinity state of b(1)-adrenoceptors were determined, using the xamoterol-induced tachycardia in pithed rats and [(3)H]CGP 12177 binding to rat cerebrocortical membranes. The respective "apparent pA(2) values" in the functional experiments were 7.9, 8.1, 5.4, 8.4, 5.7, 7.3 and 6.8 and the pK(i) values in the binding experiments were 8.8, 8.4, 6.9, 8.5, 6.7, 8.4 and 8.2. In conclusion, (-)-bupranolol and its fluorine analogue (-)-GD-6 are equipotent at the low-affinity state of beta(1)-adrenoceptors. The stereoselectivity of GD-6 and DZ-13 suggests that the low-affinity state is indeed a receptor.

Role of endocannabinoids in cardiovascular shock.

Endocannabinoids (e.g. anandamide, 2-arachidonoylglycerol or virodhamine) regulate the function of the cardiovascular system mainly in the following way: 1) by acting via CB(1) receptors, 2) by activation of CB(2) receptors, and 3) by modifying the function of vanilloid TRPV1, serotonin 5-HT(3) and alpha(7)-subunit-containing nicotinic acetylcholine receptors. Endocannabinoids are implicated in the pathogenesis of hypertension and of hypotension associated with haemorrhagic, endotoxic, and cardiogenic shock, and with advanced liver cirrhosis. There is also evidence for their involvement in the control of atherosclerosis.