sq-29548 and acetovanillone

Treatment of human pulmonary artery smooth muscle cells (HPASMCs) with the thromboxane A2 receptor antagonist, SQ29548 inhibited U46619 stimulation of phospholipase D (PLD) and NADPH oxidase activities in the cell membrane. Pretreatment with apocynin inhibited U46619 induced increase in NADPH oxidase activity. The cell membrane contains predominantly PLD2 along with PLD1 isoforms of PLD. Pretreatment with pharmacological and genetic inhibitors of PLD2, but not PLD1, attenuated U46619 stimulation of NADPH oxidase activity. U46619 stimulation of PLD and NADPH oxidase activities were insensitive to BFA and Clostridium botulinum C3 toxin; however, pretreatment with secinH3 inhibited U46619 induced increase in PLD and NADPH oxidase activities suggesting a major role of cytohesin in U46619-induced increase in PLD and NADPH oxidase activities. Arf-1, Arf-6, cytohesin-1 and cytohesin-2 were observed in the cytosolic fraction, but only Arf-6 and cytohesin-1 were translocated to the cell membrane upon treatment with U46619. Coimmunoprecipitation study showed association of Arf-6 with cytohesin-1 in the cell membrane fraction. In vitro binding of GTPγS with Arf-6 required the presence of cytohesin-1 and that occurs in BFA insensitive manner. Overall, BFA insensitive Arf6-cytohesin1 signaling axis plays a pivotal role in U46619-mediated activation of PLD leading to stimulation of NADPH oxidase activity in HPASMCs.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Acetophenones; ADP Ribose Transferases; ADP-Ribosylation Factor 6; ADP-Ribosylation Factors; Antioxidants; Botulinum Toxins; Brefeldin A; Bridged Bicyclo Compounds, Heterocyclic; Cell Membrane; Fatty Acids, Unsaturated; Gene Expression Regulation; GTPase-Activating Proteins; Guanine Nucleotide Exchange Factors; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Hydrazines; Myocytes, Smooth Muscle; NADPH Oxidases; Phospholipase D; Primary Cell Culture; Protein Synthesis Inhibitors; Pulmonary Artery; Receptors, Thromboxane A2, Prostaglandin H2; Signal Transduction; Triazoles; Vasoconstrictor Agents

BAY 41-2272 (5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine) relaxes mesenteric arteries (MA) in a synergistic fashion with nitric oxide (NO). We hypothesized that the relaxation to BAY 41-2272 is decreased in spontaneously hypertensive rats (SHR) because of the reduced NO bioavailability in this strain and that relaxation would be improved by inhibiting the oxidative stress. We aimed to evaluate the influence of oxidative stress in BAY 41-2272-induced vasorelaxation in isolated MA from SHR.. MA function was evaluated by concentration-response curves to BAY 41-2272. We measured protein expression of endothelial NO synthase (eNOS), soluble guanylyl cyclase (sGC) and human-antigen R (HuR) (sGC mRNA-stabilizing protein), sGC activity and plasma levels of superoxide dismutase (SOD), and total antioxidant status (TAS).. Cyclic guanosine monophosphate (cGMP)-dependent and -independent relaxation induced by BAY 41-2272 (0.0001-1 micromol/l) was impaired in SHR compared with Wistar-Kyoto (WKY). We observed reduced expression of eNOS, sGC and HuR, and decreased sGC activity in SHR. Plasma levels of SOD and TAS were also diminished in SHR. Incubation with SOD or indomethacin increased relaxation to BAY 41-2272 in SHR. Furthermore, acetylcholine (ACh)-induced relaxation was increased in the presence of BAY 41-2272 or SOD, apocynin, or indomethacin.. Augmented oxidative stress in SHR impaired cGMP-dependent and -independent relaxation induced by BAY 41-2272, by decreasing NO bioavailability and sGC expression and by increasing contractile activity. Inhibiton of oxidative stress improved the relaxation of BAY 41-2272 in SHR. BAY 41-2272 might be an alternative therapeutic tool for hypertension if administrated with antioxidant compounds.

Topics: Acetophenones; Acetylcholine; Animals; Antioxidants; Bridged Bicyclo Compounds, Heterocyclic; Cyclic GMP; Endothelium, Vascular; Enzyme Activation; Fatty Acids, Unsaturated; Guanylate Cyclase; Hydrazines; Nitric Oxide Synthase Type III; Nitroprusside; Oxidative Stress; Pyrazoles; Pyridines; Rats; Rats, Inbred SHR; Receptors, Cytoplasmic and Nuclear; Soluble Guanylyl Cyclase; Superoxide Dismutase; Vasodilation; Vasodilator Agents