Ethanol effects on nitric oxide production in cerebral pial cultures

Background: Although alcohol abusers are known to have higher incidences of hemorrhagic cerebrovascular diseases, it is not known whether these change s 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 c ortical 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 measu rement of nitrite, as an indication for NO release, and lactate dehydrogena se (LDH), as an index of cell membrane integrity. In addition, immunocytoch emical 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 consi sted of interleukin-1 beta (IL-1 beta; 250 pg/mL) and interferon-gamma (IFN gamma, 2 ng/mL) or to EtOH (50 to 100 mM) for 24 to 48 hr significantly el evated NO production. NO production could be attenuated by N-nitro-L-argini ne (N-arg), a nonspecific NOS inhibitor, or aminoguanidine (AG), an iNOS in hibitor. 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 a s LDH release. However, this increase was not observed in pial culture alon e or in mixed cortical culture. Nevertheless, inhibition of NO production w ith N-arg or AG did not alter the EtOH-induced LDH release in the pial cell s cocultured with cortical neurons. Conclusion: These results show that EtOH exposure led to increased producti on of NO in primary pial cell culture. In mixed culture that contained cort ical neurons and pial cells, EtOH induced increase in NO as well as LDH rel ease, which is an indication of loss of cell membrane integrity. However, E tOH-mediated LDH release in mixed cortical pial cultures was not a conseque nce 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.