u-0126 and 2--5--dideoxyadenosine

We previously reported that PEGylated conjugated linoleic acid (PCLA) as a pro-drug treatment of cultures of 3T3-L1 cells containing differentiated adipocytes caused de-differentiation by downregulation of PPARgamma2-induced adipogenesis, and cell apoptosis induced by PCLA was lower than that induced by conjugated linoleic acid (CLA) owing to the biocompatible and hydrophilic properties of poly(ethylene glycol) (PEG). To further investigate our previous observations, the present study is designed to evaluate the lipolytic action of PCLA and its role in biochemical signaling pathways of 3T3-L1 cells when compared to the CLA itself. Although both CLA and PCLA stimulated lipolysis, our results indicated a sensitivity difference between CLA and PCLA treatment: a time-dependent effect on lipolysis and p-extracellular signal-related kinases (ERK) expression was observed for PCLA-treated, but not for CLA-treated cultures. Also, the induction by PCLA of mitogen-activated protein kinase kinase (MEK)/ERK mitogen-activated protein kinase (MAPK) activation was linked to secretion of adipo-cytokines, interleukin-6 (IL-6), and interleukin-8 (IL-8), in time-dependent manners. Interestingly, adenylyl cyclase inhibitor, 2', 5'-dideoxyadenosine (DDA), pre-treatment did not prevent PCLA-stimulated lipolysis. In fact, isoproterenol, but not PCLA, caused a significant increase in cyclic adenosine monophosphate (cAMP) levels, suggesting that the PCLA-induced lipolysis was not mediated in the conventional cAMP-dependent pathway and the cAMP was the intracellular mediator for isoproterenol-induced lipolysis. Overall, our findings provide support for a role for PCLA as a pro-drug in the regulation of metabolism in adipose tissue.

Topics: 3T3-L1 Cells; Adenylyl Cyclase Inhibitors; Adipocytes; Adipokines; Animals; Butadienes; Carbon Radioisotopes; Cell Differentiation; Cells, Cultured; Cyclic AMP; Dideoxyadenosine; Enzyme Activation; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Fluorescein-5-isothiocyanate; Fluorescent Antibody Technique, Indirect; Fluorescent Dyes; Glycerol; Isoproterenol; Linoleic Acids, Conjugated; Lipolysis; Mice; Mitogen-Activated Protein Kinases; Molecular Weight; Nitriles; Oleic Acid; Polyethylene Glycols; Rhodamines; Signal Transduction; Time Factors

Vascular smooth muscle cell proliferation and migration play an important role in the pathophysiology of several vascular diseases, including atherosclerosis. Prostaglandins that have been implicated in this process are synthesized by two isoforms of cyclooxygenase (COX), with the expression of the regulated COX-2 isoform increased in atherosclerotic plaques. Bradykinin (BK), a vasoactive peptide increased in inflammation, induces the formation of prostaglandins through specific receptor activation. We hypothesized that BK plays an important role in the regulation of COX-2, contributing to the increase in production of prostaglandins in vascular smooth muscle cells. Herein we examined the signaling pathways that participate in the BK regulation of COX-2 protein levels in primary cultured aortic vascular smooth muscle cells. We observed an increase in COX-2 protein levels induced by BK that was maximal at 24 h. This increase was blocked by a B2 kinin receptor antagonist but not a B1 receptor antagonist, suggesting that the B2 receptor is involved in this pathway. In addition, we conclude that the activation of mitogen-activated protein kinases p42/p44, protein kinase C, and nitric oxide synthase is necessary for the increase in COX-2 levels induced by BK because either of the specific inhibitors for these enzymes blocked the effect of BK. Using a similar approach, we further demonstrated that reactive oxygen species and cAMP were not mediators on this pathway. These results suggest that BK activates several intracellular pathways that act in combination to increase COX-2 protein levels. This study suggests a role for BK on the evolution of the atheromatous plaque by virtue of controlling the levels of COX-2.

Topics: Adenylyl Cyclase Inhibitors; Animals; Aorta; Bradykinin; Bradykinin B2 Receptor Antagonists; Butadienes; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; Cyclooxygenase 2; Dideoxyadenosine; Enzyme Induction; Imidazoles; Immunohistochemistry; Isoquinolines; Male; Mitogen-Activated Protein Kinase Kinases; Muscle, Smooth, Vascular; NG-Nitroarginine Methyl Ester; Nitriles; p38 Mitogen-Activated Protein Kinases; Protein Kinase C; Pyridines; Rats; Rats, Sprague-Dawley; Receptor, Bradykinin B2; Sulfonamides