The activity of nSMase2 is optimal at neutral pH, is dependent on magnesium, and is enhanced by unsaturated fatty acids and anionic phospholipids, especially cardiolipin and phosphatidylserine (Marchesini et al., 2003). GW4869 was identified as an nSMase2 inhibitor both in vitro and in vivo and has been widely used to evaluate N-SMase roles in cellular response and physiological function. Unlike nSMase1, nSMase2 possesses in vivo SMase activity with overexpressing nSMase2 in MCF-7 cells showing a 40% decrease in SM levels and a concomitant 60% increase in ceramide levels (Marchesini et al., 2003); this was also observed in primary hepatocytes (Karakashian et al., 2004). Unlike the ubiquitous nSMase1, expression of nSMase2 was predominantly detected in the brain at both the protein and mRNA levels (Hofmann et al., 2000). The subcellular localization of nSMase2 has been observed in several organelles. While nSMase2 was initially localized to the Golgi in several cell lines (Hofmann et al., 2000), subsequent studies have found localization of nSMase2 predominantly at the PM. Furthermore, this PM localization could be enhanced upon stimulation (e.g. TNF-α, H2O2) or in confluent
