ucn-1028-c and arachidonyltrifluoromethane

Nitric oxide (NO) reduces the molecular activity of Na+-K+-ATPase in opossum kidney (OK) cells, a proximal tubule cell line. In the present study, we investigated the cellular mechanisms for the inhibitory effect of NO on Na+-K+-ATPase. Sodium nitroprusside (SNP), a NO donor, inhibited Na+-K+-ATPase in OK cells, but not in LLC-PK1 cells, another proximal tubule cell line. Similarly, phorbol 12-myristate 13-acetate, a protein kinase C (PKC) activator, inhibited Na+-K+-ATPase in OK, but not in LLC-PK1, cells. PKC inhibitors staurosporine or calphostin C, but not the protein kinase G inhibitor KT-5823, abolished the inhibitory effect of NO on Na+-K+-ATPase in OK cells. Immunoblotting demonstrated that treatment with NO donors caused significant translocation of PKCalpha from cytosolic to particulate fractions in OK, but not in LLC-PK1, cells. Furthermore, the translocation of PKCalpha in OK cells was attenuated by either the phospholipase C inhibitor U-73122 or the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one. U-73122 also blunted the inhibitory effect of SNP on Na+-K+-ATPase in OK cells. The phospholipase A2 inhibitor AACOCF3 did not blunt the inhibitory effect of SNP on Na+-K+-ATPase in OK cells. AACOCF3 alone, however, also decreased Na+-K+-ATPase activity in OK cells. In conclusion, our results demonstrate that NO activates PKCalpha in OK, but not in LLC-PK1, cells. The activation of PKCalpha in OK cells by NO is associated with inhibition of Na+-K+-ATPase.

Topics: Alkaloids; Animals; Arachidonic Acids; Carbazoles; Cell Line; Enzyme Activation; Enzyme Inhibitors; Estrenes; Indoles; Isoenzymes; Kidney; Kidney Tubules, Proximal; Kinetics; Naphthalenes; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Opossums; Protein Kinase C; Protein Kinase C-alpha; Pyrrolidinones; Sodium-Potassium-Exchanging ATPase; Staurosporine; Tetradecanoylphorbol Acetate

Compelling evidence indicates that activation of the JNK/SAPK signaling pathway is obligatory for apoptosis induction by multiple cell stresses that activate the sphingomyelin cycle. Moreover, ectopic expression of bcl-2 can impair apoptosis signaling by most of the cell stresses that activate the ceramide/JNK pathway. Here we show that enforced expression of bcl-2 protects prostate carcinoma cells against the induction of apoptosis by exogenous C2-ceramide. Moreover, enforced bcl-2 expression blocked the capacity of C2-ceramide to activate JNK1, indicating bcl-2 functions at the level of JNK1 or upstream of JNK1 in the ceramide/JNK pathway. The contribution of bcl2 to the regulation of the arachidonate pathway for prostate carcinoma cell survival was also investigated using highly selective inhibitors of arachidonate metabolism. Our results indicate bcl-2 can protect cells against diminished availability of arachidonic acid, 12-HETE, and 15-HETE. Finally, arachidonic acid substantially suppresses the induction of apoptosis by C2-ceramide, providing evidence for the opposing influences of these lipid signaling pathways in the mediation of prostate carcinoma cell survival. These results provide evidence for opposing influences of the ceramide and arachidonate signaling pathways in the mediation of cell death and cell survival, respectively, in prostate carcinoma cells and suggest a dual role for bcl-2 in this context.

Topics: Animals; Apoptosis; Arachidonic Acid; Arachidonic Acids; Calcium-Calmodulin-Dependent Protein Kinases; Cell Survival; Enzyme Activation; Enzyme Inhibitors; Humans; Hydroxyeicosatetraenoic Acids; JNK Mitogen-Activated Protein Kinases; Lipid Metabolism; Male; Mitogen-Activated Protein Kinases; Naphthalenes; Prostatic Neoplasms; Protein Kinase C; Proto-Oncogene Proteins c-bcl-2; Rats; Signal Transduction; Sphingosine; Transfection; Tumor Cells, Cultured