Two plasma membrane proteins, the Na+/Ca2+ exchanger (NCX) and the Ca2+-ATPase, are major regulators of free intraneuronal Ca2+ levels as they are responsible for extrusion of Ca2+ from the intracellular to the extracellular medium. Because disruption of cellular Ca2+ regulation plays a role in damage occurring under conditions of oxidative stress, studies were conducted to assess the sensitivity of the NCX to reactive oxygen species (ROS). Exchanger activity in brain synaptic plasma membranes and in transfected CHO-K1 cells was inhibited following brief exposure to the peroxyl radical generating azo initiator 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH) and to peroxynitrite. Incubation with hydrogen peroxide did not alter NCX activity, even at 800 microM concentration. In CHO-K1 cells transiently transfected with the NCX1 isoform of the exchanger, AAPH treatment decreased the maximal transport capacity (Vmax), whereas the K(act) remained unchanged. Peroxynitrite led to an increase in K(act) with no change in Vmax. Loss of activity following exposure to either AAPH or peroxynitrite was associated with the formation of high molecular weight aggregates of NCX, and AAPH also caused fragmentation of the exchanger protein. These findings suggest that the NCX is sensitive to biologically relevant ROS and could be involved in the loss of Ca2+ homeostasis observed under oxidative stress.

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Using the yeast artificial chromosome (YAC) 116 flanking the autosomal recessive spinal muscular atrophy (SMA) gene region, we have screened a human fetal brain cDNA library and isolated the cDNA clone 14-3/9 with an insert size of 2.5 kb. The cDNA clone could be identified as part of the human rRNA gene coding for 28S rRNA with a total size of 5025 bp. The human 28S rRNA is involved in the organization of the 60S ribosomal subparticle and is arranged in a 13-kb pre-rRNA transcription unit that occurs in tandem repeat clusters. Multiple copies of the rRNA gene have been mapped by pulsed field blot hybridization in the YAC contig between YAC 66 and YAC 116, which encompasses the SMA candidate gene, and additionally in the distally localized YAC 153.