Ethanol Effects on Nitric Oxide Production in Cerebral Pial Cultures

Abstract

Background: Although alcohol abusers are known to have higher incidences of hemorrhagic cerebrovascular diseases, it is not known whether these changes are associated with ethanol (EtOH) action on nitric oxide (NO) production in the cerebrovascular cells. The purpose of this study was to examine the effects of EtOH treatment on basal and cytokine-induced NO production in cortical pial cultures. Methods: Cell cultures for this study included murine primary pial vascular cells, primary glial cells and cortical neurons. These cells were exposed to cytokines or EtOH for 24 to 48 hr. The culture media were used for measurement of nitrite, as an indication for NO release, and lactate dehydrogenase (LDH), as an index of cell membrane integrity. In addition, immunocytochemical determinations were carried out to identify cell types and to assess inducible nitric oxide synthase (iNOS). Results: Exposure of primary pial vascular cultures to cytokines that consisted of interleukin-1β (IL-1β; 250 pg/mL) and interferon-γ (IFNγ; 2 ng/mL) or to EtOH (50 to 100 mM) for 24 to 48 hr significantly elevated NO production. NO production could be attenuated by N-nitro-L-arginine (N-arg), a nonspecific NOS inhibitor, or aminoguanidine (AG), an iNOS inhibitor. Increased iNOS immunoreactivity was observed in cytokines- or EtOH-treated pial cells. When pial cells were cocultured with cortical neurons, prolonged EtOH exposure led to a large increase in NO production as well as LDH release. However, this increase was not observed in pial culture alone or in mixed cortical culture. Nevertheless, inhibition of NO production with N-arg or AG did not alter the EtOH-induced LDH release in the pial cells cocultured with cortical neurons. Conclusion: These results show that EtOH exposure led to increased production of NO in primary pial cell culture. In mixed culture that contained cortical neurons and pial cells, EtOH induced increase in NO as well as LDH release, which is an indication of loss of cell membrane integrity. However, EtOH-mediated LDH release in mixed cortical pial cultures was not a consequence of the increase in NO production by these cells. Studies that use mixed cortical-pial cultures may provide a unique in vitro system for examining the interactions among glial cells, neurons, and cerebrovascular cells.