Mechanisms of apoptosis in secretory and neuronal cells : role of oxidative stress and calcium overload

Sammanfattning: Apoptosis is a widespread physiological mechanism to regulate tissue homeostasis bothduring development and in adult organs. However, the cell deletion program can beinappropriately activated or suppressed under pathological conditions. The present project wasdesigned to study the role of apoptosis in the toxicity caused by oxidative stress and calciumoverload. The effects of the free radical nitric oxide (NO ) were studied in a pancreatic beta-cell line(RlNm5F). The NO-releasing compound sodiumnitroprusside or interleukin-lbeta (IL-Ibeta)induced endogenous NO-production, stimulated inhibitory auto-ADP-ribosylation of theglycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). At later time-pointsRlNm5F cells exposed to NO underwent apoptosis. Inhibition of the nitric oxide synthaseactivity by NG-monomethyl-L-arginine prevented IL-1beta-induced NO generation and apoptoticcell killing. DNA-damage, caused by irradiation or free radicals, can stimulate expression of thetumor suppressor gene p53. The p53 protein is believed to produce growth arrest in G1-S whichallows DNA-repair. However, such conditions have also been shown to favor the occurrence ofapoptosis. We detected accumulation of the p53 protein prior to onset of apoptosis both inRINm5F cells and RAW 264.7 macrophages. To further characterize the mechanisms involved in the deletion of pancreatic cellsduring oxidative stress we used 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). We found thatDMNQ, depending on the dose, induced cell proliferation, apoptosis or necrosis. Cellproliferation was associated with induction of enzymes involved in intracellular polyaminesynthesis (i.e. ornithine decarboxylase, ODC and S-adenosyl-L-methionine decarboxylase,SAMDC). Conversely, prior to the onset of apoptosis, ODC- and SAMDC activities decreasedand cells were rapidly depleted of polyamines. Interestingly, cells were protected from apoptosiswhen incubated with the phorbol ester TPA to induce ODC and SAMDC or by supplementingspermine. Oxidative stress, calcium overload and NO-production have also been implicated inneuronal damage following ischemia. Thus, primary cultures of cerebellar granule cells (CGC)were used to further study the mechanisms of glutamate-induced cell killing. Part of the CGCpopulation exposed to glutamate rapidly lost their mitochondrial membrane potential and diedby necrosis. The surviving population recovered their mitochondrial membrane potential butlater underwent apoptosis. Nuclear lamins were degraded prior to DNA-fragmentation,indicating an early activation of proteases in cells triggered to undergo apoptosis. Furtherstudies suggested that protection from loss of mitochondrial membrane potential as well asinhibition of the phosphatase calcineurin play important roles in both necrotic and apoptoticneuronal death. In conclusion, this thesis presents evidence that apoptosis is an important determinant ofcell death both in pancreatic and neuronal cells exposed to oxidative stress or calcium overload.Onset of apoptosis was under different conditions associated with depletion of intracellularpolyamines, accumulation of p53 or degradation of nuclear larnins. Depending on the dose andduration of exposure as well as cell sensitivity, cells died by apoptosis or necrosis in bothsystems used. Neuronal apoptosis was shown to be dependent on intact mitochondria supplyingenergy, while neurons dying by necrosis rapidly lost their mitochondrial membrane potentialand were depleted of energy.Doctoral Thesis 1996 Maria AnkarcronaISBN 91-628-1867-8