Selective regulation of carboxypeptidase peptide hormone-processing enzyme during enkephalin biosynthesis in cultured bovine adrenomedullary chromaffin cells

Bovine adrenomedullary chromaffin cells in culture were incubated with reserpine or forskolin, two agents acting through different mechanisms, which increase cellular [Met]enkephalin levels by 2-fold after 72 h. Cells were harvested and chromaffin granules were purified on a linear sucrose gradient. After reserpine treatment, carboxypeptidase-processing enzyme specific activity in chromaffin granule fractions was stimulated 1.9-fold, and Co2+-stimulated carboxypeptidase specific activity was stimulated 3-fold. The increase in enzyme activity was dependent on the time of reserpine treatment. Forskolin, on the other hand, had no significant effect on carboxypeptidase activity. The differential effects of reserpine and forskolin suggest that the carboxypeptidase-processing enzyme may be selectively regulated during periods of elevated enkephalin formation. Kinetic studies revealed that in cells exposed to reserpine, the Km value for [Met]enkephalin-Arg6 for the Co2+-stimulated carboxypeptidase activity was lowered to 0.136 from 0.447 mM, but there was no change in the Km values of the non-Co2+-stimulated carboxypeptidase activity from reserpine and control groups. Cellular levels of immunoreactive carboxypeptidase-processing enzyme, measured by a radioimmunoassay method, were not altered after reserpine treatment. These data suggest that while the total number of carboxypeptidase enzyme molecules remained constant, there may be a conversion of existing enzyme molecules to a more active form which displays a higher affinity for [Met]enkephalin-Arg6 in the presence of Co2+.     

The cellular prion protein and its role in Alzheimer disease

The cellular prion protein (PrP^C) is a membrane-bound glycoprotein especially abundant in the central nervous system (CNS). The scrapie prion protein (PrP^Sc, also termed prions) is responsible of transmissible spongiform encephalopathies (TSE), a group of neurodegenerative diseases which affect humans and other mammal species, although the presence of PrP^C is needed for the establishment and further evolution of prions.The present work compares the expression and localization of PrP^C between healthy human brains and those suffering from Alzheimer disease (AD).In both situations we have observed a rostrocaudal decrease in the amount of PrP^C within the CNS, both by immunoblotting and immunohistochemistry techniques. PrP^C is higher expressed in our control brains than in AD cases. There was a neuronal loss and astogliosis in our AD cases. There was a tendency of a lesser expression of PrP^C in AD cases than in healthy ones. And in AD cases, the intensity of the expression of the unglycosylated band is higher than the di- and monoglycosylated bands.With regards to amyloid plaques, those present in AD cases were positively labeled for PrP^C, a result which is further supported by the presence of PrP^C in the amyloid plaques of a transgenic line of mice mimicking AD.The work was done according to Helsinki Declaration of 1975, and approved by the Ethics Committee of the Faculty of Medicine of the University of Navarre.Key words: cellular prion protein, Alzheimer disease, transgenic mice