Nicotine upregulates the expression of P2Y12 on vascular cells and megakaryoblasts.

BACKGROUND: P2Y12 is the major platelet receptor that mediates ADP-induced aggregation. P2Y12 is also expressed by vascular cells. The factors that regulate P2Y12 expression have not been determined. Since nicotine (NIC) has effects on platelet activation and vascular function, and because nicotinic and purinerigic receptors may interact, we determined whether nicotine altered P2Y12 expression. METHODS: Four cell lines (human coronary artery endothelial cells, HCAEC; human umbilical vein endothelial cells, HUVEC; human aortic smooth muscle cells, HASMC; and human megakaryoblastic cells, MEG-01) were cultured in the absence or presence of nicotine. Immunoblotting for P2Y12, P2Y2, and actin was performed. RESULTS: Nicotine, at concentrations of 0.1-1.0 microM, induced P2Y12 (but not P2Y2) expression in all the four cell lines. HASMC exhibited the greatest induction with a sixfold mean increase in P2Y12 expression in response to 0.25 microM nicotine. The induction was inhibited by nicotinic acetylcholine receptor antagonists. Healthy smokers were observed to have higher P2Y12 expression in platelet lysates compared to non-smokers. CONCLUSION: Nicotine induces the expression of P2Y12 in vascular cells and megakaryoblasts, and is mediated by nicotinic acetylcholine receptors. Smokers exhibit higher platelet P2Y12, possibly mediated via nicotine. These results may contribute to a better understanding of the effects of cigarette smoking on platelet activation and the vessel wall. CONDENSED ABSTRACT: The factors that regulate the expression of P2Y12, the platelet ADP receptor, have not been determined. Four cell lines (human coronary artery endothelial cells, HCAEC; human umbilical vein endothelial cells, HUVEC; human aortic smooth muscle cells, HASMC; and human megakaryoblastic cells, MEG-01) were cultured in the absence or presence of nicotine. Nicotine, at concentrations of 0.1-1.0 microM, induced P2Y12 expression in all the four cell lines. HASMC exhibited the greatest induction with a sixfold mean increase in P2Y12 expression in response to 0.25 microM nicotine. The induction was inhibited by nicotinic acetylcholine receptor antagonists. Healthy smokers were observed to have higher P2Y12 expression in platelet lysates compared to non-smokers. These results may contribute to a better understanding of the effects of cigarette smoking on platelet activation and the vessel wall.

Modulatory effect of glutathione status and antioxidants on methylmercury-induced free radical formation in primary cultures of cerebral astrocytes.

Excessive free radical formation has been implicated as one of the causative factors in neurotoxic damage associated with variety of metals, including methylmercury (MeHg). Although the mechanism(s) associated with MeHg-dependent neurotoxicity remains far from clear, overwhelming data give credence to a mediatory role for astrocytes, a major cell type that preferentially accumulates MeHg. To extend our recent findings of MeHg-induced increase in ROS formation (G. Shanker, J.L. Aschner, T. Syversen et al., Free radical formation in cerebral cortical astrocytes in culture induced by methylmercury, Mol. Brain Res. 128 (2004) 48-57), the present studies were designed to assess the effect of modulating intracellular glutathione (GSH) content, on ROS generation, in the absence and presence of MeHg. Intracellular GSH was reduced by treatment with 100 microM buthionine-L-sulfoxane (BSO) for 24 h, and increased by treatment with 1 mM l-2-oxothiazolidine-4-carboxylic acid (OTC) for 24 h. Additionally, the effects of the selective antioxidants, catalase (1000 U/ml for 1 h), an H2O2 scavenger, and n-propyl gallate (100 microM for 1 h), a superoxide radical (*O2-) and possibly hydroxyl radical (*OH) scavenger on MeHg-induced ROS formation were examined. After these treatments, astrocytes were exposed to +/-10 microM MeHg for 30 min, following which the fluorescent probes, CM-H2DCFA and CM-H2XRos were added; 20 min later, laser scanning confocal microscopy (LSCM) images were obtained. Exposure of astrocytes for 24 h to 100 microM BSO, a GSH synthesis inhibitor, led to a significant increase in mitochondrial ROS (i.e., *O2-, *NO, and ONOO-) formation, as assessed with CM-H2XRos mitotracker red dye. Similarly, BSO increased ROS formation in various intracellular organelles, as assessed with CM-H2DCFDA. BSO in combination with MeHg increased fluorescence levels in astrocytes to levels above those noted with BSO or MeHg alone, but this effect was statistically indistinguishable from either of these groups (BSO or MeHg). Pretreatment of astrocytes for 24 h with 1 mM OTC abolished the MeHg-induced increase in ROS. Results similar to those obtained with OTC were observed with the free radical scavenger, n-propyl gallate (n-PG). The latter had no significant effects on astrocytic fluorescence when administered alone. This *O2- and possibly *OH radical scavenger significantly attenuated MeHg-induced ROS formation. Catalase, an H2O2 scavenger, was less effective in reducing MeHg-induced ROS formation. Taken together, these studies point to the important protective effect of adequate intracellular GSH content as well as antioxidants against MeHg-triggered oxidative stress in primary astrocyte cultures.

Astrocyte-mediated methylmercury neurotoxicity.

Methylmercury (MeHg) is a potent neurotoxicant. Any source of environmental mercury represents a potential risk for human MeHg poisoning, because the methylation of inorganic mercury to MeHg in waterways results ultimately in its accumulation in the sea food chain, which represents the most prevalent source for human consumption. A small amount of MeHg accumulates in the central nervous system (CNS), particularly in astrocytes. Astrocytic swelling, excitatory amino acid (EAA) release and uptake inhibition, as well as EAA transporter expression inhibition are known sequelae of MeHg exposure. Herein, we review the effect of MeHg on additional transport systems (for cystine and cysteine) as well as arachidonic acid (AA) release and cytosolic phospholipase A2 (cPLA2) regulation and attempt to integrate the effects of MeHg in astrocytes within a mechanistic hypothesis that explains the inability of these cells to maintain control of the proper milieu of the extracellular fluid and, in turn, leads to neuronal demise.

Methylmercury-induced reactive oxygen species formation in neonatal cerebral astrocytic cultures is attenuated by antioxidants.

Excessive generation of reactive oxygen species (ROS) has been suggested as a causal factor in various neurodegenerative disorders, such as Parkinson's disease and Alzheimer's disease [Brain Res. 830 (1999) 10-15; Biochem. J. 310 (1995) 83-90; Free Radic. Biol. Med. 27 (1999) 612-616]. The present work examined the role of ROS in the neurotoxicity of methylmercury (MeHg). ROS formation in primary astrocytic cultures of neonatal rat cerebral cortex was monitored by 2',7'-dichlorodihydrofluorescein diacetate (H(2)DCF-DA) fluorescence. MeHg, at 10 and 20 microM caused a significant increase in ROS formation (10 microM, P<0.01; 20 microM, P<0.001). Additional studies established the effectiveness of antioxidants/free radical scavengers in attenuating the MeHg-stimulated ROS formation in the following rank-order: (1) Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a non-thiol containing antioxidant, (2) n-propyl gallate (PG), a free radical scavenger, (3) superoxide dismutase (SOD), an antioxidant enzyme that dismutates superoxide anion radical, (4) alpha-phenyl-tert-butyl nitrone (PBN), a lipophilic hydroxyl radical spin trapping agent. A significant inhibition of MeHg-induced ROS generation was also noted in astrocytes preincubated (3 h) with arachidonyl trifluoromethyl ketone (AACOCF(3,) 20 microM, P<0.05), a specific inhibitor of cytosolic phospholipase A(2) (cPLA(2)). Conversely, pretreatment (24 h) with 100 microM buthionine-L-sulfoxamine [BSO, a glutathione (GSH) synthesis inhibitor], significantly increased (P<0.05) ROS formation in MeHg treated astrocytes compared to controls. Combined, these studies invoke ROS as potent mediators of MeHg cytotoxicity and support the hypothesis that excessive ROS generation, at least in part, plays an important role in MeHg-induced neurotoxicity.

The consequences of methylmercury exposure on interactive functions between astrocytes and neurons.

Methylmercury (MeHg) is a highly neurotoxic, environmentally ubiquitous chemical that exerts its toxic effects by largely unknown mechanisms. Maintenance of optimal intracellular concentrations of glutathione (GSH) is vital for cellular defenses against damage from free radicals. Since astrocytes play an essential role in providing GSH precursors to neurons, studies were directed at the effect of MeHg on cystine transport in both cell types. Astrocytes accumulated cystine via three independent transporters, referred to as system XAG-, system XC-, and gamma-glutamyltranspeptidase (GGT). In contrast, neurons accumulated cystine exclusively via system XC- and GGT. MeHg potently inhibited cystine uptake in astrocytes (but not in neurons), and this effect could be fully accounted for by inhibition of the system XAG- transporter. The transport of glutamate in astrocytes is also inhibited by reactive oxygen species (ROS). Accordingly, additional studies examined the ability of thiol reducing or oxidizing agents to inhibit the astrocytic transport of 3H-D-aspartate, a glutamate analog. The antioxidant catalase significantly attenuated MeHg-induced inhibition of astrocytic 3H-aspartate uptake. Combinedly, these studies suggest that inhibition of cystine uptake and decreased astrocytic GSH levels and efflux reduce the availability of precursors for GSH synthesis in neurons. In addition, MeHg-induced generation of H2O2 plays a role in the inhibition of astrocytic glutamate transport. These effects likely increase neuronal vulnerability to MeHg-induced oxidative stress, and excess N-methyl D-aspartate (NMDA) receptor activation leading to neuronal demise.