Muerte encefálica y retirada del soporte

Abstract

MDCK cells were transfected with pXGH5, a plasmid containing the human growth hormone (hGH) gene, and permanently expressing cell lines were selected. Clone 3A cells, which secrete quantities of hGH through both apical and basolateral surfaces, were examined in detail. Immunofluorescence analysis using anti-hGH antibody revealed bright perinuclear staining coinciding with the area delineated by anti-p52 kDa protein (a resident Golgi protein) antibody. There appeared to be less Golgi-specific fluorescence in untransfected cells. This difference correlated with an increased amount of 52 kDa in the clone 3A cells. Morphometric analysis was performed on electron micrographs of clone 3A and untransfected cells using the fractionator to estimate average number of Golgi stacks per cell, and values were statistically analyzed. It was found that clone 3A cells contained 3.3 and untransfected cells 1.6 stacks (P < or = 0.005), respectively. When clone 3A cells were placed into defined medium, the synthesis and secretion of hGH declined 4-fold, and the number of Golgi stacks also decreased to the untransfected level within seven days. The number of Golgi stacks per untransfected cell was not affected by the presence of exogenous hGH, indicating that Golgi amplification was directly related to secretory demand. Generation times and cell volumes were identical for both cell types under all growth conditions. In addition, the kinetics of protein secretion from radiolabelled cells demonstrated that clone 3A cells generally secrete lower amounts of endogenously synthesized apical proteins than do untransfected cells, while basolateral secretion remains the same. In both cases hGH comprised only about 10% of total secretory proteins, so that the increase in total protein secretion did not seem to warrant the two-fold elaboration of Golgi by 3A cells. But there might be significant amounts of hGH which traverse the Golgi to end up in lysosomes, rather than being secreted, leading to Golgi amplification.