NA+ K+ ATPASE IMPAIRMENT AND EXPERIMENTAL GLYCATION - THE ROLE OF GLUCOSE AUTOXIDATION/

Non enzymatic glycation could be involved in the early impairment of N a+/K+ ATPase that occurs in sciatic nerve of diabetic rats. In fact, d ecrease of Na+/K+ ATPase activity is one of the first alterations show ed in experimental diabetic neuropathy. In this respect, it is known t hat in the presence of transition metals under physiological condition s, glucose can autoxidize yielding hydrogen peroxide (H2O2) and free r adical intermediates, which, in tun, inhibit the cation pump. Our expe riments were designed to determine if glucose autoxidation has any rel evance in the early steps of Na+/K+ ATPase experimental glycation. Com pared experiments with and without the sodium borohydride (NaBH4) redu ction step demonstrated that incubation of brain Na+/K+ ATPase with gl ucose 6-phosphate (G 6-P) and trace metals induced a significant decre ase in enzyme activity dramatically enhanced by addition of copper (Cu 2+). A concomitant production of H2O2 was noticed. The presence of die thylenetriaminepentaacetic acid (DTPA), a strong metal chelator, compl etely prevented Na+/K+ ATPase impairment and hydrogen-peroxide formati on. No gross structural and conformational alterations of the enzyme c an be demonstrated by intrinsic and extrinsic fluorescence measurement s. Our results suggest that during the exposure of brain Na+/K+ ATPase to glucose 6-phosphate in vitro (experimental glycation), the decreas e in activity can be correlated, at least in the early phases, to meta l-catalyzed production of oxidative species, such as H2O2, through the glucose autoxidation process, and not to glucose attachment to the en zyme. Since plasma hydroperoxides and copper appear to be elevated in diabetic patients with complications, our data suggest a critical role for oxidative reactions in the pathophysiology of the chronic complic ations of diabetes like neuropathy.