GTP Depletion Research Articles

GTP depletion and other erythrocyte abnormalities in inherited pnp deficiency

GTP levels were low and NAD + levels high in purine nucleoside phosphorylase (PNP) deficient erythrocytes, in addition to the raised deoxy-GTP (dGTP) levels previously noted by others. dGTP was also identified in the PNP deficient child's lymphocytes. A further novel finding was the conversion of hypoxanthine to inosine by the PNP deficient red cells, as compared to inosine monophosphate (IMP) in controls. This has been attributed to IMP formation with subsequent breakdown, and raises interesting questions regarding the controls which normally maintain erythrocyte nucleotide pools. These findings may also explain the gross purine overproduction seen in this defect; they may likewise be related to the associated immunodeficiency, anaemia, and other clinical manifestations. The results may also have important implications for the development and clinical use of PNP inhibitors.

Guanine nucleotide depletion and toxicity in mouse T lymphoma (S-49) cells.

Incubation of mouse T lymphoma (S-49) cells with the inosinate dehydrogenase inhibitor mycophenolic acid produced a depletion of both GTP and dGTP, and resulted in growth inhibition, partial reduction in RNA synthesis, and drastic inhibition of DNA synthesis. Similar results suggested to others that the depletion of dGTP is primarily responsible for toxicity. However, guanosine was as effective as deoxyguanosine at preventing mycophenolic acid toxicity although deoxyguanosine was more effective at elevating dGTP levels. Moreover, in hypoxanthine-guanine phosphoribosyltransferase-deficient mutants of S-49 (6MPR-3-3) deoxyguanosine was unable to prevent mycophenolic acid toxicity or to re-establish normal DNA synthesis, although it returned cellular dGTP but not GTP levels to normal. No other nucleotide levels changed in a way which could account for the toxicity. Incubation of cells with a combination of deoxyadenosine, deoxycytidine, and erythro-9-(2-hydroxy-3-nonyl)adenine produced a selective depletion of dGTP to levels similar to that produced by mycophenolic acid, but did not affect cell growth. Studies with cells synchronized by centrifugal elutriation show that the toxicity of mycophenolic acid is specific to the S-phase of the cell cycle. Addition of actinomycin D at a concentration that inhibited RNA synthesis increased the availability of GTP and re-established normal DNA synthesis in mycophenolic acid-treated S-49 cells. These results suggest that the depletion of GTP rather than that of dGTP produces toxic effects in S-49 cells and that GTP is required for DNA synthesis.

ATP mediates rapid reversal of cyclic GMP phosphodiesterase activation in visual receptor membranes.

Weak or strong lights will activate visual receptor rod disk membrane (RDM) cyclic GMP phosphodiesterase (PDE) in the presence of GTP cofactor. A similarly activated GTPase can exhaust small amounts of initially present GTP to deactivate the PDE. However, further additions of GTP reactivate PDE without more light, and deactivation by simple GTP depletion takes minutes or more, even at GTP concentrations 100 to 1,000 times lower than physiological levels. A more rapid deactivation mechanism must exist if modulation of cytoplasmic cyclic GMP by light is to play a role on the time scale (seconds) of events in vision. We report here that ATP is essential to such rapid control and that its presence permits multiple cycles of activation-deactivation. The complete control mechanism seems to involve gamma phosphate transfer from both ATP and GTP.

Reduction in β‐Adrenergic Response of Cultured Glioma Cells Following Depletion of Intracellular GTP

1. When C6 glioma cells were incubated with mycophenolic acid, a potent and specific inhibitor of IMP:NAD oxidoreductase (EC 1.2.1.14) there was a marked depletion of the cellular content of GTP. The viability of the cells was unaffected. 2. The adenosine 3':5'-monophosphate (cyclic AMP) response of C6 glioma cells to the beta-adrenergic stimulant, (+/-)isoprenaline, was considerably reduced after treatment with mycophenolic acid. The diminished response to (+/-)isoprenaline was prevented by the inclusion of guanine in the culture medium along with mycophenolic acid. 3. The adenylate cyclase response to (+/-)isoprenaline of whole homogenates from C6 cells treated with mycophenolic acid was also depressed; the response was restored to normal by the addition of GTP. 4. The adenylate cyclase response to (+/-)isoprenaline of a membrane fraction prepared from homogenates of C6 cells was almost totally dependent on the presence of added GTP. Membrane fractions from control and mycophenolic-acid-treated C6 cells gave similar adenylate cyclase responses to (+/-)isoprenaline in the presence of GTP. 5. It is concluded that mycophenolic acid may depress the beta-adrenergic sensitivity of C6 cells by depleting the cellular content of GTP.