cyclic-gmp and phorbol-12-13-didecanoate

Vasodilation by agents such as bradykinin and ATP is dependent on nitric oxide, the endothelium-dependent relaxing factor (EDRF). The release of EDRF results in elevation of cGMP in endothelial and smooth muscle cells (9). The signaling pathway that leads to increases in cGMP is not completely understood. The role of protein kinase C (PKC) in the elevation of cGMP induced by ATP and bradykinin was studied in cultured porcine aortic endothelial cells, by measuring PKC phosphorylation of a substrate and by measuring cGMP levels by radioimmunoassay. Extracellular ATP and bradykinin simultaneously elevated cGMP levels and PKC activity. The PKC inhibitors staurosporine, calphostin C, and Cremophor EL (T. Tamaoki and H. Nakano. Bio/Technology 8: 732-735, 1990; F. K. Zhao, L. F. Chuang, M. Israel, and R. Y. Chuang. Biochem. Biophys. Res. Commun. 159: 1359-1367, 1989) prevented the elevation of cGMP elicited by ATP and reduced that produced by bradykinin. Cremophor did not affect the elevation of cGMP by nitroprusside, an agent that directly increases guanylate cyclase activity (9). The PKC activator phorbol 12-myristate 13-acetate, but not a phorbol ester analog inactive on PKC, also elevated cGMP levels. These results suggest that EDRF agonists elevate cGMP in endothelial cells via PKC stimulation.

Topics: Adenosine Triphosphate; Animals; Aorta, Thoracic; Bradykinin; Cells, Cultured; Cyclic GMP; Endothelium, Vascular; Enzyme Activation; Enzyme Inhibitors; Fetus; Kinetics; Naphthalenes; NG-Nitroarginine Methyl Ester; Nitric Oxide; Phorbol Esters; Polyethylene Glycols; Protein Kinase C; Staurosporine; Swine; Tetradecanoylphorbol Acetate; Time Factors; Vasodilator Agents

The incubation of primary cultures of rat hepatocytes with lipopolysaccharide (LPS) or biologically active phorbol esters promotes the release of nitric oxide to the incubation medium. This process is the result of the induction of the Ca(2+)-and calmodulin-independent form of nitric oxide synthase. Both the release of nitric oxide to the incubation medium and the expression of nitric oxide synthase activity exhibited a lag period of about 45-60 min after cell stimulation. Exposure of hepatocytes to both stimuli produced an antagonistic effect on nitric oxide release, with a half-maximal inhibition obtained with 14 nM phorbol 12,13-dibutyrate at saturating concentration of LPS. Incubation of cells with alpha-phorbol 12,13-didecanoate failed to counteract the effect of LPS or to induce nitric oxide synthase, suggesting that activation of protein kinase C was involved in this process.

Topics: Amino Acid Oxidoreductases; Animals; Arginine; Calcimycin; Carcinogens; Cells, Cultured; Cyclic GMP; Diglycerides; Enzyme Induction; Kinetics; Lipopolysaccharides; Liver; Nitric Oxide Synthase; Phorbol 12,13-Dibutyrate; Phorbol Esters; Rats; Tetradecanoylphorbol Acetate

The regulation of T4 5'-deiodinase activity was studied in cultured GH4C1 cells. Enzyme activity was measured in cell sonicates as the release of radioiodide from [125I]T4. Enzyme activity was stimulated 2- to 3-fold by hypothyroid serum and activators of protein kinase C, such as TRH and phorbol esters. The hypothyroid serum effect was maximal by 3 h, whereas TRH and phorbol esters required 6 h to achieve a maximal effect. The hypothyroid serum effect was gone within 4 h of returning the cells to control medium. In contrast, the TRH and phorbol ester effects persisted 24-48 h after removal of those agents. Both T4 and rT3 were at least as potent as T3 in blocking the effect of hypothyroid serum. The stimulation of 5'-deiodinase induced by hypothyroid serum was additive with that induced by kinase C activators. Trifluoperazine blocked the effect of TRH and phorbol esters, but not that of hypothyroid serum. It is concluded that stimulation of 5'-deiodinase activity can occur by at least two independent mechanisms: one involving hypothyroidism and another involving activation of protein kinase C. The relative potencies of various iodothyronines for abolishing the hypothyroid effect differ markedly from the relative binding affinities of these agents for the nuclear T3 receptor, suggesting that this thyroid hormone effect may not be mediated by the classical nuclear thyroid hormone receptor.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Calcimycin; Cell Line; Colforsin; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Activation; Hypothyroidism; Iodide Peroxidase; Phorbol Esters; Pituitary Neoplasms; Protein Kinase C; Rats; Tetradecanoylphorbol Acetate; Thyroid Hormones; Thyrotropin-Releasing Hormone; Thyroxine; Trifluoperazine; Triiodothyronine; Triiodothyronine, Reverse

Topics: Animals; Carbachol; Cell Line; Cyclic GMP; Inositol Phosphates; Mice; Neuroblastoma; Phorbol Esters; Phorbols; Protein Kinase C; Receptors, Muscarinic; Sugar Phosphates; Tetradecanoylphorbol Acetate