CELL-DEATH, SURVIVAL AND PROLIFERATION IN TETRAHYMENA-THERMOPHILA EFFECTS OF INSULIN, SODIUM-NITROPRUSSIDE, 8-BROMO CYCLIC-GMP, N-G-METHYL-L-ARGININE AND METHYLENE-BLUE

Cells of the ciliate Tetrahymena thermophila produce compounds that ac t as autocrine (paracrine) survival and/or growth factors. 8-Bromo cyc lic GMP, sodium nitroprusside, hemin, protoporphyrin IX, human recombi nant and bovine insulin were tested for their ability to substitute fo r the cell-produced factors and stimulate cell survival and proliferat ion. The cells were inoculated into conical flasks in a nutritionally complete, chemically defined medium at known cell densities from 5 to 5000 cells/ml. In unsupplemented medium cells at 5 to 500 cells/ml ('l ow initial cell density cultures') died within 8 h, whereas cells at 1 000 and 5000 cells/ml ('high initial cell density cultures') prolifera ted with lag phases lasting for up to 4 h. In the presence of insulin compounds, hemin, protoporphyrin IX, or 8-bromo cyclic GMP, cells also proliferated at all low initial cell densities. Sodium nitroprusside was effective over two separate concentration ranges: at the nanomolar levels as well at low pico- to femtomolar levels. At initial populati on densities of up to 50 cells/ml the cells at both concentrations of sodium nitroprusside survived about 4-fold longer than the controls. A t 500 initial cells/ml, cells at the high concentrations of sodium nit roprusside survived about 4-fold longer than those of the control cult ures; they proliferated in the low concentrations of sodium nitropruss ide. Concentrations of hemin, too low to have any effects on their own , had synergistic effects with sodium nitroprusside. N-G-methyl-L-argi nine inhibited proliferation at high initial cell densities. This inhi bitory action was reduced by high concentrations of L-arginine, protop orphyrin IX, sodium nitroprusside, or 8-bromo cGMP, but not by insulin . Methylene blue inhibited cell proliferation at high initial cell den sities. This inhibition was circumvented by addition of 8-bromo cCMP. The findings that insulin-related material may be released from Tetrah ymena and that insulin and sodium nitroprusside increase intracellular cGMP in these cells are discussed in relation to the presented result s. Together these observations suggest that cGMP is responsible for su pporting cell survival in Tetrahymena and switching the cells into the ir proliferative mode and that cell-produced signal molecules and insu lin stimulate an NO-dependent guanylate cyclase into producing cGMP. ( C) 1996 Academic Press Limited