ATP- and carbachol -stimulated 1,2-diacylglycerol production during sensitization of adenylyl cyclase

Stimulation of LM5 cells with the phorbol ester 4$\beta$-phorbol 12-myristate 13-acetate (PMA), causes a 2-4 fold sensitization of hormonally-stimulated adenylyl cyclase (AC) activity. This effect is thought to be due to protein kinase C (PKC)-mediated phosphorylation of either G$\sb{\rm i}$ or the catalytic subunit of AC. PKC are components of the phosphatidylinositol-4,5-bisphosphate phospholipase C (PIP$\sb2$-PLC) pathway. The currently accepted model of this pathway is that its activation by an agonist results in the production of inositol 1,4,5-triphosphate (IP$\sb3$) which causes Ca$\sp{++}$ mobilization, and 1,2-diacylglycerols (DAG) which activate PKC. Based on this model, we predicted that stimulation of purinergic and muscarinic receptors with the agonists ATP and carbachol (CCh), respectively in the LM5 cells, should sensitize AC. Surprisingly we found that only stimulation of the purinergic receptors in these cells caused a sensitization of PGE$\sb1$-stimulated AC measured in cell-free assays.^ We hypothesized that ATP-and CCh-stimulated differential DAG production contributes to the effectiveness of these two agonists to sensitize PGE$\sb1$-stimulated AC activity. To test this hypothesis directly, we performed a combined high-performance liquid chromatography and gas-liquid chromatography analysis of the DAG produced in the LM5 cells in response to stimulation with ATP and CCh.^ We found that both ATP and CCh increased levels of 23 species of DAG. Relative to the control levels (0.261 nmol DAG/100 nmol phospholipid) the CCh-induced increase in DAG levels was 280% (0.738 $\pm$ 0.051 nmol DAG/100 nmol phospholipid) whereas the ATP-induced levels increased 180% (0.441 t 0.006 nmol DAG/100 nmol phospholipid). Neither agonist created new species or eliminated the existing ones. The major species which comprised $\approx$50% of the total cellular DAG in all of the groups were 16:0-18:1, 18:0-18:1, 18:1-18:1, and 18:0-20:4. CCh was more effective than ATP at stimulating these major DAG species.^ It is concluded that factor(s) other than DAG contribute(s) to the differences between ATP-and CCh-sensitization of PGE$\sb1$-stimulated AC activity in the LM5 cells. ^

Metabolism and actions of 2 -chloro-2$\sp\prime$-fluoro-arabinosyladenine

2-Chloro-9-(2-deoxy-2-fluoro-$\beta $-D-arabinofuranosyl)adenine(Cl-F-ara-A) is a new deoxyadenosine analogue which is resistant to phosphorolytic cleavage and deamination, and exhibits therapeutic activity for both leukemia and solid tumors in experimental systems. To characterize its mechanism of cytotoxicity, the present study investigated the cellular pharmacology and the biochemical and molecular mechanisms of action of Cl-F-ara-A, from entrance of the drug into the cell, chemical changes to active metabolites, targeting on different cellular enzymes, to final programmed cell death response to the drug treatment.^ Cl-F-ara-A exhibited potent inhibitory action on DNA synthesis in a concentration-dependent and irreversible manner. The mono-, di-, and triphosphates of Cl-F-ara-A accumulated in cells, and their elimination was non-linear with a prolonged terminal phase, which resulted in prolonged dNTP depression. Ribonucleotide reductase activity was inversely correlated with the cellular Cl-F-ara-ATP level, and the inhibition of the reductase was saturated at higher cellular Cl-F-ara-ATP concentrations. The sustained inhibition of ribonucleotide reductase and the consequent depletion of deoxynucleotide triphosphate pools result in a cellular Cl-F-ara-ATP to dATP ratio which favors analogue incorporation into DNA.^ Incubation of CCRF-CEM cells with Cl-F-ara-A resulted in the incorporation of Cl-F-ara-AMP into DNA. A much lesser amount was associated with RNA, suggesting that Cl-F-ara-A is a more DNA-directed compound. The site of Cl-F-ara-AMP in DNA was related to the ratio of the cellular concentrations of the analogue triphosphate and the natural substrate dATP. Clonogenicity assays showed a strong inverse correlation between cell survival and Cl-F-ara-AMP incorporation into DNA, suggesting that the incorporation of Cl-F-ara-A monophosphate into DNA is critical for the cytotoxicity of Cl-F-ara-A.^ Cl-F-ara-ATP competed with dATP for incorporation into the A-site of the extending DNA strand catalyzed by both DNA polymerase $\alpha$ and $\varepsilon$. The incorporation of Cl-F-ara-AMP into DNA resulted in termination of DNA strand elongation, with the most pronounced effect being observed at Cl-F-ara-ATP:dATP ratio $>$1. The presence of Cl-F-ara-AMP at the 3$\sp\prime$-terminus of DNA also resulted in an increased incidence of nucleotide misincorporation in the following nucleotide position. The DNA termination and the nucleotide misincorporation induced by the incorporation of Cl-F-ara-AMP into DNA may contribute to the cytotoxicity of Cl-F-ara-A. ^